Methods and compositions for size-controlled homopolymer tailing of substrate polynucleotides by a nucleic acid polymerase

ABSTRACT

The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3′ end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/898,460, filed Feb. 17, 2018, now issued as U.S. Pat. No.10,731,194, which is a divisional of U.S. Non-Provisional applicationSer. No. 14/384,113, filed Sep. 9, 2014, now issued as U.S. Pat. No.9,896,709, which is a U.S. National Phase of International ApplicationNo. PCT/US2013/031104, filed Mar. 13, 2013, which claims the prioritybenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/610,296, filed Mar. 13, 2012, and U.S. Provisional Application No.61/613,784, filed Mar. 21, 2012, the disclosures of each of which areincorporated herein by reference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 6, 2017, isnamed 17-21004-US_SL.txt and is 27,358 bytes in size.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions for addingtails of specific lengths to a substrate polynucleotide. The inventionalso contemplates methods and compositions for immobilization of tailedsubstrates to a solid support.

BACKGROUND OF THE INVENTION

Many current next-generation sequencing (NGS) platforms require specialDNA and RNA preparations prior to sequencing. Most commonly usedpreparations involve addition of adaptor sequences to the ends ofdouble-stranded DNA fragments through a ligation reaction. The reactiontypically involves blunt-ended DNA or DNA with a single deoxyadenosine(dA) nucleotide at the 3′ end and a high concentration of DNA ligase,and the reaction results in formation of a significant number ofchimeric templates. Template-independent polymerases such asDNA-specific terminal deoxynucleotidyl transferase (TdT), andRNA-specific poly(A) and poly(U) polymerases potentially represent anattractive alternative approach for preparation of DNA and RNA for NGSanalysis with the challenging caveat that the length of polymeric tailsproduced by these enzymes varies in a wide range (from 20 to 500nucleotides), depends on many factors, and is not easy to control, thusreducing their utility for NGS.

SUMMARY OF THE INVENTION

Accordingly, the present disclosure provides a composition comprising anucleic acid polymerase and an attenuator molecule. The disclosurecontemplates that the attenuator molecule is any biomolecule thatassociates with a tail sequence added to a substrate polynucleotide andcontrols the addition of a tail sequence to the 3′ end of the substratepolynucleotide. The sequence that is added to the substratepolynucleotide is referred to herein as a tail sequence, or simply atail, and the process of adding a nucleotide to a substratepolynucleotide is referred to herein as tailing. An attenuator molecule,as used herein, is a polynucleotide, a polypeptide, a polysaccharide,and combinations thereof. In aspects where the attenuator molecule is apolynucleotide, it is further contemplated that the polynucleotide is acircular molecule, or that the polynucleotide comprises a peptidenucleic acid, a Schizophyllan polysaccharide, a locked nucleic acid andcombinations thereof.

As described above, the attenuator molecule associates with a tailsequence added to the substrate polynucleotide and controls the additionof nucleotides thereto. In some embodiments, the attenuator molecule isa polynucleotide that hybridizes to a sequence added to a substratepolynucleotide, wherein the number of nucleotides added to the substratepolynucleotide is essentially equal to the number of nucleotides in theportion of the attenuator molecule that associates with the tailsequence. In some aspects, the number of nucleotides added to thesubstrate polynucleotide is essentially equal to a multiple of thenumber of nucleotides in the attenuator molecule that associates withthe tail sequence. As used herein, the terms “essentially” and“essentially equal” are understood to mean approximately orapproximately equal.

In some embodiments, the nucleic acid polymerase is atemplate-independent polymerase. In one aspect, the nucleic acidpolymerase is a DNA polymerase, and in a further aspect the DNApolymerase is terminal deoxynucleotidyl transferase (TdT). In relatedembodiments, the nucleic acid polymerase is a RNA polymerase, which invarious aspects is selected from the group consisting of poly(A)polymerase, RNA-specific nucleotidyl transferase and poly(U) polymerase.

It is contemplated by the disclosure that, in some embodiments, theattenuator molecule comprises a nucleotide selected from the groupconsisting of 2′-deoxythymidine 5′¬monophosphate (dTMP),2′-deoxyguanosine 5′-monophosphate (dGMP), 2′-deoxyadenosine5′¬monophosphate (dAMP), 2′-deoxycytidine 5′-monophosphate (dCMP),2′-deoxyuridine 5′¬monophosphate (dUMP), thymidine monophosphate (TMP),guano sine monophosphate (GMP), adenosine monophosphate (AMP), cytidinemonophosphate (CMP), uridine monophosphate (UMP), a base analog, andcombinations thereof. Thus, in certain embodiments the attenuatormolecule comprises an attenuator sequence that is a heteropolymericsequence or a homopolymeric sequence, wherein the sequence is either adinucleotide sequence or a homopolymer sequence.

In various aspects, the attenuator molecule comprises 1 nucleotide, 2nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 10nucleotides, 20 nucleotides, 30 nucleotides, 50 nucleotides, 100nucleotides or more.

The attenuator molecule, in some embodiments, comprises a blocking groupand in one aspect, the blocking group is on the 3′ end of the molecule.The blocking group prevents extension of the attenuator molecule by thenucleic acid polymerase. Thus, in various aspects the blocking group isselected from the group consisting of at least one ribonucleotide, atleast one deoxynucleotide, a C3 spacer, a phosphate, adideoxynucleotide, an amino group, and an inverted deoxythymidine.

In some embodiments of the disclosure, the attenuator molecule furthercomprises an adaptor sequence, an identifier tag sequence, or both,located 5′ to the attenuator sequence homopolymer sequence ordinucleotide sequence. hi one embodiment, the attenuator molecule isimmobilized. In further embodiments, a ligase is present in thecomposition. In another embodiment, the composition further comprises aligase enzyme.

In another embodiment, the attenuator molecule is an attenuator-adaptormolecule which comprises an attenuator sequence and further comprises asequence W positioned adjacent the attenuator sequence and iscomplementary to a sequence X on a separate polynucleotide; thecomposition further comprising an adaptor molecule comprising a sequenceY complementary to a sequence V, wherein sequence V is the same lengthas Y or is less than the same length as sequence Y, the adaptor moleculebeing a separate molecule from the attenuator-adaptor molecule.

In some embodiments of the disclosure, the attenuator molecule furthercomprises a next generation sequencing (NGS) adaptor sequence. An NGSadaptor sequence differs from an adaptor sequence in that the NGSadaptor sequence is useful in a sequencing platform. In some embodimentsof the disclosure, the attenuator molecule further comprises a nextgeneration sequencing (NGS) adaptor sequence comprises sequence X andsequence Y (for further description of the various sequences (e.g.,sequence X, sequence Y, etc.) discussed herein, see Figures anddiscussion, below), an identifier tag sequence, or a combinationthereof, located 5′ to a homopolymer sequence or dinucleotide sequence.In embodiments wherein the attenuator molecule further comprises anadaptor sequence, it is referred to herein as an attenuator-adaptormolecule. In one embodiment, the attenuator molecule and/orattenuator-adaptor molecule is immobilized. In related embodiments,sequence X and sequence Y are NGS adaptor sequences that are compatiblewith Illumina, Ion Torrent, Roche 454 or SOLiD sequencing platforms.

In further embodiments, sequence X and sequence Y are on separateattenuator-adaptor molecules, while in other embodiments sequence X andsequence Y are adjacent to each other on the same attenuator-adaptormolecule.

The disclosure also provides, in some embodiments, compositions whereinan adaptor sequence further comprises a cleavable sequence (Z) that islocated between sequence X and sequence Y. In some aspects, the cleavagesite in sequence Z is at least one dU base or RNA base, or a restrictionendonuclease site.

Also provided by the disclosure are compositions wherein the attenuatormolecule is single stranded or is at least partially double stranded. By“partially double stranded” is meant that the attenuator moleculecomprises a single stranded portion and a double stranded portion. Insome aspects wherein the attenuator molecule is at least partiallydouble stranded, the partially double stranded attenuator molecule isproduced by annealing a portion of the attenuator molecule to an adaptormolecule (which comprises an adaptor sequence that, in some embodiments,is an NGS adaptor sequence) to which it is complementary. In someaspects, annealing is (a) between an attenuator molecule and a separateadaptor molecule, or (b) annealing occurs in a single attenuatormolecule that forms a hairpin structure, thus the attenuator moleculecomprises both a homopolymeric sequence or dinucleotide sequence and anadaptor sequence.

In still further embodiments, the attenuator molecule comprises asequence W that is fully complementary to adaptor sequence X. In someaspects, sequence W is also all or partially complementary to adaptorsequence Y.

In various aspects, the attenuator molecule comprises a homopolymericsequence selected from the group consisting of poly (dA), poly (dT),poly (dC), poly (dG), poly (dU), poly (rA), poly (U), poly (rC), poly(rG) and a heteropolymeric, or a dinucleotide, sequence comprisingcombinations of: (i) dA and rA bases, (ii) dT, dU and U bases, (iii) dCand rC bases, or (iv) dG and rG bases.

In further aspects, the attenuator molecule comprisesdeoxyribonucleotides and is degradable with a DNA-specific nuclease. Insome of these aspects, the DNA-specific nuclease is DNase I. In furtherembodiments, a composition provided by the disclosure comprises a singlestrand circularization ligase, including but not limited to CircLigaseand/or CircLigase II.

The disclosure also provides, in some aspects, a composition thatcomprises a DNA polymerase which lacks proofreading activity, and KapaHiFi Polymerase, which possesses proofreading activity. In additionalembodiments, a composition of the disclosure comprises a ligase enzyme.

Further embodiments of the disclosure provide a composition thatcomprises a restriction endonuclease capable of cleaving sequence Z,which is located between sequence X and sequence Y and comprises arestriction endonuclease site, when hybridized to a complementary X′Z′Y′polynucleotide.

In some embodiments, a composition is provided wherein a partiallydouble stranded adaptor sequence comprised of sequence V and sequence Ywherein V is a truncated complement of Y and comprises a blocked 3′ end,such that the partially double stranded adaptor can be blunt ligated toa double stranded substrate molecule. In another embodiment, sequence Vis fully complementary to sequence Y.

The disclosure further contemplates compositions wherein the attenuatormolecule is degradable. In some aspects, the attenuator moleculecomprises dU bases and is degradable by incubation with a dU-glycosylase(which creates abasic sites) followed by incubation at a temperaturethat is above 80° C. (introduces breaks within abasic sites), or amixture of dU-glycosylase and an apurinic/apyrimidinic endonuclease.Thus, the disclosure provides compositions, in various aspects, whereinthe attenuator molecule comprises dU bases and incubation with adU-glycosylase destabilizes the attenuator molecule, or incubation witha dU¬glycosylase and subsequent incubation at a temperature that isabove 80° C. degrades the attenuator molecule, or the attenuatormolecule is incubated with a mixture of dU-glycosylase and anapurinic/apyrimidinic endonuclease. In further aspects, the attenuatormolecule comprises a ribonucleotide and is degradable with aribonuclease under conditions sufficient for ribonuclease activity. Inrelated aspects, the ribonuclease is selected from the group consistingof RNase H, RNase HII, RNase A, and RNase T1.

In further aspects, the attenuator molecule comprisesdeoxyribonucleotides and is degradable with a DNA-specific nuclease. Insome of these aspects, the DNA-specific nuclease is DNase I.

The disclosure also provides a method of extending a substratepolynucleotide comprising incubating the substrate polynucleotide with acomposition as described herein under conditions sufficient to allowaddition of a tail sequence to the 3′ end of the substratepolynucleotide, and wherein the addition of the tail sequence allowsassociation between the tail sequence and the attenuator molecule toform a complex. In another aspect, the method further comprisesdegrading the attenuator molecule following extension of the substratepolynucleotide. In a further aspect, the method further comprisesisolating the extended substrate polynucleotide. Other aspects of themethods further comprise mixing a composition as described herein withthe substrate polynucleotide and a nucleotide that is complementary tothe homopolymeric portion of the attenuator molecule.

According to various aspects of the disclosure, the substratepolynucleotide is a single stranded polynucleotide or is a doublestranded polynucleotide. The double stranded polynucleotide, in someaspects, has a blunt end, a 3′ overhanging end, a 3′ recessed end, or afree 3′ hydroxyl group. The present disclosure provides methods whereinthe substrate polynucleotide is double stranded, and in certain aspects,the double stranded substrate polynucleotide is produced by annealing afirst substrate polynucleotide to a second substrate polynucleotideunder conditions sufficient to allow the first substrate polynucleotideto associate with the second substrate polynucleotide. According tofurther aspects of the disclosure, the substrate polynucleotidecomprises a free 3′ hydroxyl group. The single stranded polynucleotide,in various embodiments, is prepared by denaturation of fragmented doublestranded DNA or from reverse transcription of RNA. The double strandedpolynucleotide, in some aspects, has a blunt end or a 3′ overhanging endwith a free 3′ hydroxyl group.

In various aspects of the methods of the disclosure, a multiplicity ofnucleotides are added to the substrate polynucleotide. The number ofnucleotides added to the substrate polynucleotide comprises, in variousaspects, at least about 1 nucleotide and up to about 10, 20, 50 or 100nucleotides, at least about 3 nucleotides and up to about 10, 20, 50 or100 nucleotides, at least about 10 nucleotides and up to about 20, 30,50 or 100 nucleotides, at least about 5 nucleotides and up to about 10,20, 50 or 100 nucleotides, at least about 10 nucleotides and up to about20, 30, 50 or 100 nucleotides, at least about 1 nucleotide and up toabout 5, 10, or 20 nucleotides, at least about 3 nucleotides and up toabout 5, 10, or 20 nucleotides, at least about 5 nucleotides and up toabout 20, 40 or 50 nucleotides, at least 1 nucleotide, at least 2nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5nucleotides, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, at least 68, at least 69, at least 70, atleast 71, at least 72, at least 73, at least 74, at least 75, at least76, at least 77, at least 78, at least 79, at least 80, at least 81, atleast 82, at least 83, at least 84, at least 85, at least 86, at least87, at least 88, at least 89, at least 90, at least 91, at least 92, atleast 93, at least 94, at least 95, at least 96, at least 97, at least98, at least 99, at least 100 nucleotides or more.

Further embodiments provided by the disclosure include those wherein theattenuator molecule associates with the tail sequence over all or aportion of the attenuator molecule length. In some embodiments, theattenuator molecule associates with the tail sequence during the processof adding the tail sequence. Association of the attenuator molecule tothe tail sequence, in further aspects, regulates addition of nucleotidesto the substrate polynucleotide. The attenuator molecule additionallycomprises an adaptor sequence, in some aspects, and the adaptor sequenceis ligated by a ligase enzyme to the substrate polynucleotide duringaddition of the tail sequence to the substrate polynucleotide, and invarious embodiments the ligase is a DNA ligase or a RNA ligase.

It is also contemplated by the disclosure that the conditions of themethod, in some aspects, regulate addition of the tail sequence to thesubstrate polynucleotide. With respect to the conditions that arecontemplated to regulate addition of the tail sequence to the substratepolynucleotide, it is contemplated that, in some aspects, the additionof the tail sequence to the substrate polynucleotide is temperaturesensitive. Conditions wherein the temperature is between about 4° C. andabout 50° C. are contemplated. In aspects wherein a thermostablepolymerase is used, the temperature can be above 50° C. Accordingly, infurther aspects the temperature is between about 50° C. and about 90° C.

In another embodiment, the addition of the tail sequence to thesubstrate polynucleotide is time sensitive, and in various aspects theincubation step is allowed to progress for a length of time in the rangeof about 0.5 minutes to about 120 minutes. In further embodiments, theaddition of the tail sequence to the substrate polynucleotide is pHsensitive. In some of these embodiments, the addition of the tailsequence to the substrate polynucleotide is performed under conditionswherein pH is in the range of about pH 5.0 to about pH 9.0.

In various embodiments, the substrate polynucleotide is DNA or RNA.

Methods provided herein also include those wherein theattenuator-adaptor molecule is immobilized. In some aspects, theimmobilized attenuator-adaptor molecule is ligated by a DNA or RNAligase to a substrate polynucleotide during addition of a tail sequenceto the substrate polynucleotide resulting in immobilization of thesubstrate polynucleotide. In further aspects, the amount of ligaseenzyme added to a reaction is from about 0.1 to about 1000 units (U).

In certain aspects, the methods described herein further comprisemagnesium in an amount of about 1 mM to about 100 mM. In furtheraspects, the methods further comprise potassium or sodium in an amountof about 1 mM to about 1 M.

In a specific aspect of the disclosure, a method of extending a DNAsubstrate polynucleotide is provided comprising mixing the DNA substratepolynucleotide with TdT enzyme, a degradable attenuator polynucleotidecomprising a 3′ phosphate and nucleotides that are complementary to thehomopolymeric portion of the attenuator polynucleotide, incubating themixture at 37° C. for about 30 minutes, followed by an additionalincubation at 70° C. for about 10 minutes, degrading the attenuatormolecule by adding a DNA glycosylase and incubating the mixture at 37°C. for about 5 minutes, and optionally isolating the extended DNAsubstrate polynucleotide.

In another aspect, the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising mixing a substrate polynucleotidewith TdT enzyme, an attenuator polynucleotide comprising tworibonucleotides at the 3′ end, and nucleotides that are complementary tothe homopolymeric portion of the attenuator polynucleotide, incubatingthe mixture at 30° C. for 30 minutes, followed by an additionalincubation at 70° C. for about 10 minutes to inactivate the TdT enzyme,and optionally isolating the extended DNA substrate polynucleotide.

The disclosure further provides, in one aspect, a method of extending asubstrate RNA polynucleotide comprising mixing the substrate RNApolynucleotide with an RNA polymerase, a degradable attenuatorpolynucleotide and ribonucleotides that are complementary to thehomopolymeric portion of the attenuator polynucleotide, incubating themixture at 30° C. for about 30 minutes, followed by an additionalincubation at 95° C. for about 10 minutes, degrading the attenuatormolecule by adding a DNA glycosylase and incubating the mixture at 37°C. for about 10 minutes, and optionally isolating the extended substratepolynucleotide.

In another aspect, the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising annealing attenuator and adaptormolecules that are partially complementary to each other by heating amixture of the attenuator and the adaptor molecules in a suitable bufferto about 100° C. and then cooling to about 25° C., wherein the annealingresults in a partially double stranded attenuator-adaptor molecule,mixing the DNA substrate polynucleotide with TdT enzyme, a ligaseenzyme, the partially double stranded attenuator¬adaptor molecule andnucleotides that are complementary to the homopolymeric portion of theattenuator-adaptor molecule, incubating at about 37° C. for about 15 toabout 30 minutes, and optionally isolating the extended DNA substratepolynucleotide ligated to the attenuator-adaptor molecule.

The disclosure further provides, in one aspect, a method of extending aDNA substrate polynucleotide comprising annealing one attenuator-adaptormolecule to a second, biotinylated adaptor molecule that are at leastpartially complementary to each other by heating a mixture of the twomolecules in a suitable buffer to about 100° C. and then cooling toabout 25° C., wherein the annealing results in a double strandedbiotinylated attenuator-adaptor molecule, immobilizing the doublestranded biotinylated attenuator-adaptor molecule by mixing the doublestranded biotinylated attenuator-adaptor molecule with a solutioncomprising a streptavidin-coated magnetic bead at about 25° C. for about30 to about 60 minutes, resulting in immobilization of the doublestranded biotinylated attenuator-adaptor molecule to thestreptavidin-coated magnetic bead, incubating the immobilized doublestranded biotinylated attenuator-adaptor molecule attached to thestreptavidin-coated magnetic bead with the DNA substrate polynucleotide,TdT enzyme, a ligase enzyme and nucleotides that are complementary tothe homopolymeric portion of the double stranded attenuator molecule atabout 37° C. for 15 to about 30 minutes, washing the solution with NaOHto remove non-biotinylated single stranded DNA from the beads, andoptionally isolating the extended DNA substrate polynucleotide ligatedto the double stranded biotinylated attenuator-adaptor molecule.

In another aspect, a method of extending a RNA substrate polynucleotideis provided comprising annealing an attenuator molecule and an adaptormolecule that are at least partially complementary to each other byheating a mixture of the two molecules in a suitable buffer to about100° C. and then cooling to about 25° C., wherein the annealing resultsin a partially double stranded attenuator-adaptor molecule; mixing anRNA substrate polynucleotide with poly (A) or poly(U) enzyme, a ligaseenzyme, the partially double stranded attenuator-adaptor molecule andribonucleotides that are complementary to the single-strandedhomopolymeric portion of the partially double stranded attenuatormolecule; incubating at about 30° C. to about 37° C. for about 15-30minutes; and optionally isolating the extended RNA substratepolynucleotide ligated to the attenuator-adaptor molecule.

In a further aspect, the disclosure provides a method of extending andimmobilizing an RNA substrate polynucleotide comprising annealing anattenuator-adaptor molecule to a biotinylated adaptor molecule that areat least partially complementary to each other by heating a mixture ofthe two molecules in a suitable buffer to about 100° C. and then coolingto about 25° C., wherein the annealing results in a partially doublestranded biotinylated attenuator-adaptor molecule; immobilizing thepartially double stranded biotinylated attenuator-adaptor molecule bymixing the partially double stranded biotinylated attenuator-adaptormolecule with a solution comprising a streptavidin-coated magnetic beadat about 25° C. for about two hours, resulting in immobilization of thepartially double stranded biotinylated attenuator-adaptor molecule tothe streptavidin-coated magnetic bead; incubating the immobilizedpartially double stranded biotinylated attenuator-adaptor moleculeattached to the streptavidin-coated magnetic bead with the RNA substratepolynucleotide, poly(A) or poly(U) polymerase, a ligase enzyme andribonucleotides that are complementary to the single strandedhomopolymeric portion of the partially double strandedattenuator-adaptor molecule at about 30° C. to about 37° C. for about15-30 minutes; washing the solution with NaOH to remove non-biotinylatedsingle stranded polynucleotide from the beads; and optionally isolatingthe extended and immobilized RNA substrate polynucleotide ligated to thedouble stranded biotinylated attenuator-adaptor molecule.

The disclosure also provides, in various embodiments, methods wherein aDNA polymerase and dNTPs are mixed to perform a polymerase extension ofa substrate polynucleotide, said polymerase extension occurringsubsequent to controlled homopolymer tailing and leading toincorporation of NGS adaptor sequence(s) sequence X, sequence Y orsequences X and Y 3′ to the substrate homopolymer that are complementaryto the additional sequence X′ and sequence Y′ that are 5′ to thehomopolymer of the attenuator molecule.

In further embodiments, NGS adaptor sequence X, sequence Y or sequencesX and Y are optionally ligated by a ligase enzyme to the substratepolynucleotide during addition of nucleotides to the substratepolynucleotide. In other embodiments, NGS adaptor sequence X, sequence Yor sequences X and Y are optionally ligated by a ligase enzyme to thesubstrate polynucleotide after addition of nucleotides to the substratepolynucleotide. In related embodiments, the ligase is a DNA ligase or aRNA ligase.

In still further embodiments, an attenuator molecule sequence W isoptionally truncated with respect to sequences X′ and Y′ to allow afull-length X′Y′ polynucleotide primer to displace the truncatedattenuator and enable polymerase extension to create a double strandedadapted substrate molecule. As used herein, an “adapted molecule” is asubstrate molecule that has undergone a tailing and ligation reaction.

The disclosure further provides embodiments wherein the substratemolecule, following homopolymer addition and polymerase extension, isoptionally incubated with a single stranded DNA circularization ligasethat results in circularization of the adapted single stranded DNAmolecule. In one embodiment, circularization of the attenuator moleculecomprising sequence X and sequence Y is prevented by degradation.

In another embodiment, the substrate molecule, following homopolymeraddition and ligation, is optionally incubated with a single strandedDNA circularization ligase which results in circularization of theadapted single stranded DNA molecule.

In a further embodiment of the disclosure, circularization of the XZYadaptor molecule is prevented by formation of a double-stranded orpartially double-stranded attenuator-adaptor molecule.

Embodiments of the disclosure contemplate cleavage of the circular DNAmolecule at sequence Z, said cleavage resulting from incubation with,for example and without limitation, dU glycosylase and anapurinic/apyrimidinic endonuclease in embodiments wherein Z comprises dUbases.

Further embodiments of the disclosure include cleavage of the circularDNA molecule at sequence Z, said cleavage resulting from incubation withRNase H, RNase H II (in embodiments wherein Z comprises RNA bases) or bya restriction enzyme, following hybridization of an oligonucleotidecomplementary to the XZY junction.

In further aspects, the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising: mixing the DNA substratepolynucleotide with a polymerase enzyme and a ligase enzyme, an adaptorpolynucleotide comprising NGS adaptor sequences X and Y and cleavablesequence Z, wherein the adaptor optionally comprises a 3′ ribonucleotideand a 5′ phosphate, and is annealed to an attenuator with a truncatedNGS adaptor sequence W and 3′ block, and deoxynucleotides that arecomplementary to the homopolymeric portion of the attenuatorpolynucleotide; performing the tailing and ligation simultaneousreaction, heat inactivating the polymerase and ligase enzymes, followedby incubation with a single strand specific circularization ligaseenzyme; optionally including a cleavage reaction at the Z sequence or anamplification reaction with reverse X and Y primers, either of which isperformed to resolve the circular molecule into a completed linear NGSlibrary molecule.

In another aspect, the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising mixing a DNA substratepolynucleotide with a polymerase enzyme, an attenuator with NGS adaptorsequences X′ and Y′ and comprising a 3′ extension block and a cleavagesite as described herein, and deoxynucleotides that are complementary tothe homopolymeric portion of the attenuator polynucleotide; performingthe tailing reaction, heat inactivating the polymerase enzyme, followedby addition of a DNA polymerase and dNTP mix to perform a polymeraseextension to the tailed substrate polynucleotide to incorporate NGSadaptor sequences X and Y 3′ of the homopolymer addition on thesubstrate; the attenuator¬template polynucleotide is then degraded withRNase or dU-glycosylase, followed by incubation with a single strandspecific circularization ligase enzyme; optionally including a cleavagereaction at a Z sequence or an amplification reaction with reverse X andY primers, either of which is performed to resolve the circular moleculeinto a completed linear NGS library molecule.

In another aspect, the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising mixing the DNA substratepolynucleotide with a polymerase enzyme and a ligase enzyme, an adaptorpolynucleotide comprising the NGS adaptor sequence X, wherein theadaptor optionally comprises a 3′ extension block and a 5′ phosphate,and is annealed to an attenuator with a truncated NGS adaptor sequence Wand 3′ block, and deoxynucleotides that are complementary to thehomopolymeric portion of the attenuator polynucleotide; performing thetailing and ligation simultaneous reaction, heat inactivating thepolymerase and ligase enzymes, followed by incubation with a primercomplementary to full-length NGS adaptor sequence X′ that is capable ofdisplacing the attenuator molecule, a DNA polymerase and dNTPsoptionally including dUTP to perform an extension reaction to generatesecond strand and a double stranded substrate molecule with a blunt end;performing a ligation with T4 DNA ligase and a blunt-end adaptor whichis formed by annealing two polynucleotides comprising NGS adaptorsequence Y and a truncated complement (sequence V) and a 3′ phosphate,wherein the Y polynucleotide is ligated to the 5′ phosphate of thesubstrate molecule to complete a linear NGS library molecule; optionallythe synthesized strand is degraded using dU glycosylase and heat to 95°C.

A further aspect of the disclosure provides a method of extending a DNAsubstrate polynucleotide comprising: mixing the DNA substratepolynucleotide with a polymerase enzyme, an attenuator with NGS adaptorsequences X′ and comprising a 3′ extension block and a cleavage site,and deoxynucleotides that are complementary to a homopolymeric portionof the attenuator polynucleotide; performing a tailing reaction, heatinactivating the polymerase enzyme, followed by addition of aproofreading DNA polymerase and dNTP mix to perform a polymeraseextension to incorporate NGS adaptor sequence X 3′ of the homopolymeraddition on the substrate and to remove non-complementary bases from theattenuator 3′ end to enable polymerase extension to generate a blunt enddouble stranded substrate; performing a ligation with T4 DNA ligase anda blunt-end adaptor molecule which is formed by annealing twopolynucleotides comprising NGS adaptor sequence Y and a truncatedcomplement (sequence V) and a 3′ phosphate, where the Y polynucleotideis ligated to the 5′ phosphate of the substrate molecule to complete alinear NGS library molecule; optionally the synthesized strand isdegraded using dU glycosylase and heat to between about 90° C. and 100°C. In various embodiments, the mixture is heated to between about 90° C.and 95° C., or between about 95° C. and 100° C., or is about 90° C., orabout 91° C., or about 92° C., or about 93° C., or about 94° C., orabout 95° C., or about 96° C., or about 97° C., or about 98° C., orabout 99° C., or about 100° C.

In still another aspect, a method of extending an RNA substratepolynucleotide is provided comprising: mixing the RNA substratepolynucleotide with a Poly(A) or Poly(U) enzyme and T4 DNA ligase, a DNAadaptor polynucleotide comprising the NGS adaptor sequence X, whereinthe adaptor optionally comprises a 3′ extension block and a 5′phosphate, and is annealed to an RNA attenuator with a truncated NGSadaptor sequence W and 3′ block, and ribonucleotides that arecomplementary to the homopolymeric portion of the attenuatorpolynucleotide; performing a tailing and ligation simultaneous reaction,heat inactivating the Poly(A) or Poly(U) enzyme, followed by incubationwith a primer complementary to full-length NGS adaptor sequence X′ thatis capable of displacing the attenuator molecule truncated for sequenceX′, a reverse transcriptase and dNTPs to perform an extension reactionto generate a second strand and a double stranded substrate molecule;performing a magnetic bead DNA purification step followed by stranddenaturation at 95° C.; a second simultaneous tailing and ligation isthen performed using a polymerase enzyme and a ligase enzyme and a DNAattenuator-adaptor molecule that will add a homopolymer and Y NGSadaptor sequence to the free 3′ end of the substrate molecule, thuscompleting a linear NGS library molecule where an optional DNApurification step is required.

The disclosure also provides a method of NGS library synthesis usingcontrolled homopolymer tailing and ligation followed by circularizationof the substrate polynucleotide (FIG. 25), where fragmented singlestranded DNA is prepared by denaturation of fragmented double strandedDNA or by reverse transcription of RNA, wherein controlled tailing andligation is performed in the presence of the polymerase enzyme, a ligaseenzyme, nucleotide D (where D=dATP, dTTP or dGTP) and anattenuator-adaptor molecule, wherein the attenuator-adaptor molecule isformed by annealing two polynucleotides: polynucleotide XZY andpolynucleotide W(H)n.

Polynucleotide XZY comprises a 5′ phosphate, and a ribonucleotide baseor other blocking group at the 3′ end which prevents addition of ahomopolymer tail. Sequences X and Y represent adaptor sequences of anNGS library and optional ID tag. Optional sequence Z is used forcleavage and is comprised of a dU base, an RNA base or a restrictionendonuclease site. Polynucleotide W(H)n comprises two sequences: a 5′sequence W that is complementary to the 5′ portion of the polynucleotideXZY and a homopolymeric attenuator sequence (H), where H is A, T or Cbase, and n=10-30. Polynucleotide W(H), comprises a 3′ blocking groupincluding but not limited to a 3′ phosphate, dideoxynucleotide,C3-spacer, inverted thymidine or one or more ribonucleotides thatspecifically inhibit addition of bases by the polymerase enzyme. In thereaction, the polymerase enzyme will add a limited number of bases tothe 3′ end of DNA substrates followed by ligation of the XZY adaptormolecule by a ligase enzyme. In certain embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is betweenabout 1-50 dA or dT bases or between about 1-50 dG bases. In furtherembodiments, the number of bases added by the polymerase to the 3′ endof a DNA substrate is from about 1 nucleotide and up to about 5, 10, 20,30, 40 or 50 dA, dT or dG nucleotides, or from about 5 and up to about10, 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about 10 andup to about 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about9 to about 12 dA, dT or dG nucleotides, or from about 6 to about 8 dA,dT or dG nucleotides. In additional embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is at leastabout 1, at least about 2, at least about 3, at least about 4, at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 17, at least about 18, at least about 19, at least about20, at least about 21, at least about 22, at least about 23, at leastabout 24, at least about 25, at least about 26, at least about 27, atleast about 28, at least about 29, at least about 30, at least about 31,at least about 32, at least about 33, at least about 34, at least about35, at least about 36, at least about 37, at least about 38, at leastabout 39, at least about 40, at least about 41, at least about 42, atleast about 43, at least about 44, at least about 45, at least about 46,at least about 47, at least about 48, at least about 49 or at leastabout 50 dA, dT or dG nucleotides. In further embodiments, the number ofbases added by the polymerase to the 3′ end of a DNA substrate is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more dA, dT or dG nucleotides.

Following optional heat inactivation of the polymerase enzyme and theligase enzyme, incubation with single stranded circularization ligase(Epicentre/Illumina) results in circularization of the adapted singlestranded DNA molecule. Circularization of the XZY polynucleotide isprevented by formation of a double-stranded attenuator-adaptor complexbetween the XZY and W(H), polynucleotides. Circularized NGS librariescan be directly used for cluster formation (emulsion PCR in the case ofIon Torrent, 454 and Solid platforms, and bridge amplification in thecase of Illumina platforms) or directly for sequencing (PacBio).Optionally, cleavage of the circular DNA molecule is achieved by dUglycosylase and an apurinic/apyrimidinic endonuclease (in the case whenZ sequence comprises a dU base), by RNase H or RNase H II (in the casewhen Z sequence comprises an RNA base) or by a restriction endonuclease.In the latter two cases, hybridization of a polynucleotide complementaryto the XZY junction is necessary to provide a template for RNase H orrestriction endonuclease. Alternatively, an amplification reaction isperformed to resolve the circular to linear form.

The disclosure also provides an alternative method for NGS librarysynthesis using controlled homopolymer tailing followed by polymeraseextension and circularization (FIG. 26), wherein tailing of singlestranded substrate polynucleotides is performed in the presence of apolymerase enzyme, nucleotide D (where D=dATP, dTTP or dGTP) and anattenuator-template polynucleotide, where the attenuator-templatepolynucleotide comprises three sequences: a 5′ sequence Y′ that iscomplementary to adaptor sequence Y, sequence X′ that is complementaryto adaptor sequence X and a homopolymeric attenuator sequence (H), whereH is A, T or C base, and n=10-30. The attenuator-template polynucleotideadditionally comprises a phosphate group, ribonucleotide or otherblocking group at the 3′ end. Sequences X and Y represent adaptorsequences for an NGS library, and optional ID tag. Both the attenuatorsequence and X′,Y′ sequences have degradable bases such asribonucleotides or dU. In the presence of the attenuator molecule, apolymerase enzyme adds a limited number of bases to the 3′ end of DNA.In certain embodiments, the number of bases added by the polymerase tothe 3′ end of a DNA substrate is between about 1-50 dA or dT bases orbetween about 1-50 dG bases. In further embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is from about 1nucleotide and up to about 5, 10, 20, 30, 40 or 50 dA, dT or dGnucleotides, or from about 5 and up to about 10, 15, 20, 30, 40 or 50dA, dT or dG nucleotides, or from about 10 and up to about 12, 15, 20,30, 40 or 50 dA, dT or dG nucleotides, or from about 10 to about 13 dA,dT or dG nucleotides. In additional embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is at leastabout 1, at least about 2, at least about 3, at least about 4, at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 17, at least about 18, at least about 19, at least about20, at least about 21, at least about 22, at least about 23, at leastabout 24, at least about 25, at least about 26, at least about 27, atleast about 28, at least about 29, at least about 30, at least about 31,at least about 32, at least about 33, at least about 34, at least about35, at least about 36, at least about 37, at least about 38, at leastabout 39, at least about 40, at least about 41, at least about 42, atleast about 43, at least about 44, at least about 45, at least about 46,at least about 47, at least about 48, at least about 49 or at leastabout 50 dA, dT or dG nucleotides. In further embodiments, the number ofbases added by the polymerase to the 3′ end of a DNA substrate is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more dA, dT or dG nucleotides.

After optional heat inactivation of the polymerase enzyme and the ligaseenzyme, a DNA polymerase and dNTP mix are added to perform polymeraseextension which results in addition of X and Y sequences to the 3′ endof the homopolymeric sequence (D). Following attenuator-templatedegradation by addition of dU-glycosylase or RNase, incubation withsingle strand circularization ligase (Epicentre/Illumina) results incircularization of the adapted single stranded DNA molecule. Ifnecessary, an optional amplification is performed to resolve thecircular to linear form. Alternatively an optional sequence Z between Xand Y is used to create linear form following hybridization of acomplementary polynucleotide to the XZY domain and a cleavage reaction(as described herein above). In certain embodiments, the optionalamplification that is performed to resolve the circular to linear formis a technique including, without limitation, inverse PCR.

Also contemplated is a method of NGS library synthesis comprisingcontrolled homopolymer tailing and ligation followed by reverse strandsynthesis and blunt adaptor ligation (FIG. 27), wherein tailing andligation are performed in the presence of TdT enzyme, E. coli DNAligase, nucleotide D (where D=dATP, dTTP or dGTP) and anattenuator-adaptor molecule that is formed by annealing twopolynucleotides: polynucleotide X and polynucleotide W(H)n.Polynucleotide X comprises a 5′ phosphate and a 3′ blocking group, wheresequence X comprises an NGS library adaptor sequence and optional IDtag. Polynucleotide W(H)n consists of two sequences: a 5′ sequence Wthat is complementary to the 5′ portion of polynucleotide X and ahomopolymeric attenuator sequence (H), where H is A, T or C base, andn=10-30, and additionally comprises a 3′ blocking group. In thereaction, the polymerase enzyme adds a limited number of bases to the 3′end of DNA substrates followed by ligation of the attenuator-adaptormolecule by a ligase enzyme. In certain embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is betweenabout 1-50 dA or dT bases or between about 1-50 dG bases. In furtherembodiments, the number of bases added by the polymerase to the 3′ endof a DNA substrate is from about 1 nucleotide and up to about 5, 10, 20,30, 40 or 50 dA, dT or dG nucleotides, or from about 5 and up to about10, 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about 10 andup to about 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about9 to about 12 dA, dT or dG nucleotides, or from about 6 to about 8 dA,dT or dG nucleotides. In additional embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is at leastabout 1, at least about 2, at least about 3, at least about 4, at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 17, at least about 18, at least about 19, at least about20, at least about 21, at least about 22, at least about 23, at leastabout 24, at least about 25, at least about 26, at least about 27, atleast about 28, at least about 29, at least about 30, at least about 31,at least about 32, at least about 33, at least about 34, at least about35, at least about 36, at least about 37, at least about 38, at leastabout 39, at least about 40, at least about 41, at least about 42, atleast about 43, at least about 44, at least about 45, at least about 46,at least about 47, at least about 48, at least about 49 or at leastabout 50 dA, dT or dG nucleotides. In further embodiments, the number ofbases added by the polymerase to the 3′ end of a DNA substrate is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more dA, dT or dG nucleotides.

Following optional heat inactivation of the polymerase enzyme and theligase enzyme, addition of a DNA polymerase and primer containing 5′sequence X′, 1-10 H bases (or, in further embodiments, from about 1 toabout 5, 7, or 10 H bases, or from about 5 to about 6, 8 or 10 H bases,or from about 6 to about 8 H bases) at the 3′ end and optional dU basesleads to reverse strand replication, thus forming either adouble-stranded blunt end (in the presence of proofreading DNApolymerase) or double-stranded end with 3′ dA base (in the case when DNApolymerase lacks the proofreading activity), and wherein thepolymerization mix optionally comprises dUTP. Ligation of a blunt-end ordT-adaptor is achieved by a ligase enzyme, wherein the adaptor to beligated is formed by two polynucleotides: polynucleotide Y, where Y is asecond NGS adaptor and polynucleotide V that is complementary to the 3′portion of polynucleotide Y. The 3′ end of polynucleotide V has aphosphate blocking group. Ligation results in covalent attachment of the3′ end of polynucleotide Y to the 5′ phosphate of the original DNAfragment, whereas no ligation is formed between the 5′ end ofpolynucleotide V and the 3′ end of the primer-extension product.Optionally the replicated DNA strand and primer X′ is degraded byincubation with dU glycosylase and 95° C. incubation. In someembodiments, the incubation takes place between about 90° C. and 100° C.In further embodiments, the mixture is heated to between about 90° C.and 95° C., or between about 95° C. and 100° C., or is about 90° C., orabout 91° C., or about 92° C., or about 93° C., or about 94° C., orabout 95° C., or about 96° C., or about 97° C., or about 98° C., orabout 99° C., or about 100° C.

The disclosure also provides an alternative method for NGS libraryconstruction comprising controlled tailing and polymerization followedby reverse strand synthesis and blunt ligation (FIG. 28), whereintailing is performed in the presence of the polymerase enzyme,nucleotide D (where D=dATP, dTTP or dGTP) and attenuator-templatepolynucleotide comprising two sequences: a 5′ sequence X′ that iscomplementary to NGS adaptor X, and a homopolymeric attenuator sequence(H), where H is A, T or C base, and n=10-30, and additionally comprisesa 3′ ribonucleotide and internal degradable bases such as ribonucleotideor dU. In the presence of the attenuator molecule, the polymerase enzymeadds a limited number of bases to the 3′ end of DNA substrates andfollowing optional heat inactivation, the inclusion of a DNA polymerasewith proofreading activity and dNTP mix extends the DNA substrate toinclude sequence X and also removes excessive non-complementary basesfrom the attenuator polynucleotide 3′ terminus and then leads to reversestrand replication which results in a double-stranded blunt end. Thepolymerization mix can optionally comprise dUTP. Ligation of a blunt-endadaptor is achieved by T4 DNA ligase, where the blunt-end adaptor isformed by two polynucleotides: polynucleotide Y, where Y is a sequenceof second NGS adaptor and polynucleotide V that is complementary to the3′ portion of the polynucleotide Y, and where the 3′ end ofpolynucleotide V comprises a phosphate or other blocking group. Bluntligation results in covalent attachment of the 3′ end of polynucleotideY to the 5′ phosphate of the original DNA fragment, whereas no ligationoccurs between the 5′ end of the polynucleotide V and the 3′ end of theprimer-extension product. Optionally, the replicated DNA strand andprimer X′ is degraded by incubation with dU glycosylase and 95° C.incubation. In some embodiments, the incubation takes place betweenabout 90° C. and 100° C. In further embodiments, the mixture is heatedto between about 90° C. and 95° C., or between about 95° C. and 100° C.,or is about 90° C., or about 91° C., or about 92° C., or about 93° C.,or about 94° C., or about 95° C., or about 96° C., or about 97° C., orabout 98° C., or about 99° C., or about 100° C.

Another method of the disclosure for NGS library preparation comprisestwo sequential tailing and ligation reactions (FIG. 29), wherein thefirst tailing and ligation reaction is performed in the presence of apolymerase enzyme, a ligase enzyme, nucleotide D (where D=dATP, dTTP ordGTP) and attenuator-adaptor molecule, which is formed by annealing twopolynucleotides: polynucleotide X and polynucleotide W(H)n, wherepolynucleotide X comprises a 5′ phosphate, 3′ blocking group and NGSadaptor sequence and optional ID tag; and polynucleotide W(H)n whichcomprises two sequences: 5′ sequence W that is complementary to the 5′portion of polynucleotide X and a homopolymeric attenuator sequence (H),where H is A, T or C base, and n=10-30, and additionally comprises a 3′blocking group.

In the reaction, a polymerase enzyme adds a limited number of bases tothe 3′ end of DNA substrates (1-50 dA or dT bases and 1-50 dG bases),followed by ligation of the first attenuator-adaptor molecule by aligase enzyme. Following optional heat inactivation of the polymeraseand the ligase, addition of a primer containing a 5′ sequence X′complementary to sequence X and 1-10 H bases at the 3′ end results inprimer annealing to the adaptor sequence X and displacement of theattenuator polynucleotide. Additionally, primer X′ can comprise an rHblocking base at the 3′ end to prevent polymerase-mediated primertailing with the second tailing and ligation reaction. Addition of a DNApolymerase will extend the primer and replicate the reverse strand ofthe substrate, where the reaction is optionally stopped by EDTA andpurified by magnetic bead-based DNA purification before a secondaddition of polymerase enzyme adds a limited number of bases to the 3′end of DNA extension product (1-50 dA or dT bases and 1-50 dG bases) inthe presence of the second attenuator that is annealed to a second NGSadaptor Y, followed by ligation of the second attenuator-adaptormolecule comprising the second NGS adaptor Y by a ligase enzyme.Optionally, the polymerase and the ligase are heat inactivated, followedby magnetic bead based DNA purification. As above, and in furtherembodiments, the number of bases added by the polymerase to the 3′ endof a DNA substrate is from about 1 nucleotide and up to about 5, 10, 20,30, 40 or 50 dA, dT or dG nucleotides, or from about 5 and up to about10, 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about 10 andup to about 15, 20, 30, 40 or 50 dA, dT or dG nucleotides, or from about9 to about 12 dA, dT or dG nucleotides, or from about 6 to about 8 dA,dT or dG nucleotides. In additional embodiments, the number of basesadded by the polymerase to the 3′ end of a DNA substrate is at leastabout 1, at least about 2, at least about 3, at least about 4, at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 16,at least about 17, at least about 18, at least about 19, at least about20, at least about 21, at least about 22, at least about 23, at leastabout 24, at least about 25, at least about 26, at least about 27, atleast about 28, at least about 29, at least about 30, at least about 31,at least about 32, at least about 33, at least about 34, at least about35, at least about 36, at least about 37, at least about 38, at leastabout 39, at least about 40, at least about 41, at least about 42, atleast about 43, at least about 44, at least about 45, at least about 46,at least about 47, at least about 48, at least about 49 or at leastabout 50 dA, dT or dG nucleotides. In further embodiments, the number ofbases added by the polymerase to the 3′ end of a DNA substrate is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more dA, dT or dG nucleotides.

The disclosure also provides methods for NGS library synthesis using acombination of 5′ tailed random primer extension on a substratepolynucleotide followed by controlled tailing and ligation (FIG. 30),where intact or fragmented single stranded nucleic acid is contactedunder conditions that allow hybridization to a random primer comprisingtwo domains, a 5′ domain X where sequence X comprises in variousembodiments an NGS adaptor sequence and optional identification tag anda 3′ domain comprising a random sequence n, thereby enabling primerhybridization to any region within a nucleic acid sample, and in thepresence of a polymerase and nucleotides under appropriate reactionconditions, primer extension and incorporation of adaptor X at the 5′terminus of the extension products is achieved. The primer is variousaspects comprise a 5′ label, including but not limited to biotin.Following an optional purification or nucleotide degradation step,tailing and ligation reactions are performed on the extension product inthe presence of a polymerase, a ligase enzyme, nucleotide D (where D=A,T or G) and an attenuator-adaptor molecule that is formed by annealingtwo polynucleotides: polynucleotide Y and polynucleotide V(H).Polynucleotide Y comprises a 5′ phosphate and a 3′ blocking group, wheresequence Y comprises a second NGS library adaptor sequence and optionalidentification tag. Polynucleotide V(H), consists of two sequences: a 5′sequence V that is complementary to the 5′ portion of polynucleotide Yand a homopolymeric attenuator sequence (H)ll where H is A, T or C base,and n=10-30, and additionally comprises a 3′ blocking group. In thereaction, the polymerase enzyme adds a limited number of bases (i.e.,from about 1 to about 50 nucleotides) to the 3′ end of primer extensionproducts followed by ligation of the Y/V(H)n attenuator-adaptor moleculeby a ligase enzyme, thus completing the addition of both the first andsecond NGS adaptors.

The disclosure also contemplates a method of targeted NGS librarysynthesis using a combination of 5′ tailed target-specific primerextension on a substrate polynucleotide followed by controlled tailingand ligation (FIG. 31), where intact or fragmented single strandednucleic acid is subject to hybridization by an plurality oftarget-specific primers comprising two domains, a 5′ domain X common toall primers of the plurality where sequence X comprises an NGS adaptorsequence and optional identification tag, and a 3′ domain unique to eachprimer of the plurality, each comprising a target-specific sequence,thereby enabling primer hybridization to any desired plurality oftargets within a nucleic acid sample, and in the presence of apolymerase and nucleotides under appropriate reaction conditions, primerextension and incorporation of adaptor X at the 5′ terminus of theextension products is achieved. The primer plurality can additionallycomprise a 5′ label including but not limited to biotin. Following anoptional purification or nucleotide degradation step, tailing andligation reactions are performed in the presence of a polymerase enzyme,a ligase enzyme, nucleotide D (where D=A, T or G) and anattenuator-adaptor molecule that is formed by annealing twopolynucleotides: polynucleotide Y and polynucleotide V(H)_(n).Polynucleotide Y comprises a 5′ phosphate and a 3′ blocking group, wheresequence Y comprises a second NGS library adaptor sequence and optionalidentification tag. Polynucleotide V(H)_(n) consists of two sequences: a5′ sequence V that is complementary to the 5′ portion of polynucleotideY and a homopolymeric attenuator sequence (H)_(n) where H is A, T or Cbase, and n=10-30, and additionally comprises a 3′ blocking group. Inthe reaction, the polymerase enzyme adds a limited number of bases(i.e., from about 1 to about 50 nucleotides) to the 3′ end of primerextension products followed by ligation of the Y/V(H)_(n)attenuator-adaptor molecule by a ligase enzyme, thus completing theaddition of both NGS adaptors on each target in the plurality.

Also contemplated is a method of targeted NGS library synthesiscomprising controlled homopolymer tailing and ligation followed bytarget-specific primer extension and target-specific blunt adaptorligation (FIG. 32), wherein tailing and ligation are performed in thepresence of a polymerase, a ligase, nucleotide D (where D=A, T or G) andan attenuator-adaptor molecule that is formed by annealing twopolynucleotides: polynucleotide X and polynucleotide W(H)_(n).Polynucleotide X comprises a 5′ phosphate and a 3′ blocking group, wheresequence X comprises an NGS library adaptor sequence and optionalidentification tag. Polynucleotide W(H)_(n) comprises two sequences: a5′ sequence W that is complementary to the 5′ portion of polynucleotideX and a homopolymeric attenuator sequence (H)_(n) where H is A, T or Cbase, and n=10-30, and additionally comprises a 3′ blocking group. Inthe reaction, the polymerase adds a limited number of bases (i.e., fromabout 1 to about 50 nucleotides) to the 3′ end of nucleic acidsubstrates followed by ligation of the attenuator-adaptor molecule by aligase. Following optional heat inactivation of the polymerase andligase, addition of a polymerase, nucleotides and plurality oftarget-specific primers under appropriate reaction conditions leads toformation of either a double-stranded blunt end (in the presence ofproofreading DNA polymerase) or double-stranded end with 3′ dA base (inthe case when DNA polymerase lacks the proofreading activity) forfragments comprising a complementary sequence to the plurality oftarget-specific primers. The primer plurality can additionally comprisea 5′ label including but not limited to biotin. Ligation of a blunt-endor dT-adaptor to the plurality of target specific products is achievedby a ligase enzyme, wherein the adaptor to be ligated is formed by twopolynucleotides: polynucleotide Y, where Y is a second NGS adaptor andpolynucleotide V that is complementary to the 3′ portion ofpolynucleotide Y. The 3′ end of polynucleotide V has a phosphateblocking group. Ligation results in covalent attachment of the 3′ end ofpolynucleotide Y to the 5′ phosphate of the plurality of target-specificfragments, whereas no ligation is formed between the 5′ end ofpolynucleotide V and the 3′ end of the target specific primer-extensionproducts. Optionally the completed plurality of target-specific libraryproducts are amplified by PCR using NGS adaptor-specific primers.

In FIG. 33, a summary of target-specific NGS library preparation methodsinvolving a controlled tailing and ligation step are depicted. Method 1summarizes the method described in FIG. 32 with optional targetedlibrary bead capture in Method 2. Method 3 summarizes a method in whicha whole genome NGS library is constructed and then followed bytarget-specific primer extension and targeted library bead capture andamplification. Methods 4 and 5 depict alternate workflows to the methodpresented in FIG. 31. In Method 4, a plurality of biotinylatedtarget-specific primers extend select regions from a fragmented nucleicacid sample, followed by blunt or TA ligation of the first NGS adaptor,bead capture, then controlled tailing and ligation to add the second NGSadaptor. Method 5 is similar to 4 except that in the first steptarget-specific primers comprise 5′ tails with an NGS adaptor sequence.In Method 6, following controlled tailing and ligation to introduce afirst NGS adaptor and adaptor-specific primer extension, denaturationfollowed by second NGS adaptor 5′ tailed target-specific primerextension completes the NGS library that can be further amplified. Inany of the methods for target-specific NGS library preparation, it iscontemplated that use of the attenuator molecules described hereinenables one to multiplex. In additional embodiments, use of theattenuator molecules described herein enables one to immobilize thelibrary products to a surface.

For any of the various embodiments of whole genome or targeted libraryconstruction using the disclosed method of controlled tailing andligation to introduce an NGS adaptor sequence, a second adaptor sequenceis introduced by either a second controlled tailing and ligation or isintroduced by blunt or TA ligation to a double-stranded substratefollowing a primer extension reaction. As shown in FIG. 37, variousmethods are contemplated for blunt and TA adaptor ligation that eitherligate one strand selectively or ligates both strands. Thus, the adaptorsequence comprises, in various embodiments, a blunt end or aT-overhanging end (thus allowing TA ligation to occur). In additionalembodiments, the adaptor sequence can be blunt-ended, have a T-base3′-overhang, 5′-phosphate, or 3′-group blocking ligation (for exampleand without limitation, a dideoxynucleotide) or a combination thereof.

The disclosure further provides a method of extending a substratepolynucleotide comprising: (1) incubating a mixture comprising thesubstrate polynucleotide with (i) a polymerase enzyme; (ii) acomposition comprising an attenuator molecule comprising an attenuatorsequence and further comprises a sequence W positioned adjacent theattenuator sequence and is complementary to an adaptor sequence X on aseparate polynucleotide; the composition further comprising an adaptormolecule comprising a sequence Y complementary to a sequence V, whereinsequence V is the same length as Y or is less than the same length assequence Y, the adaptor molecule being a separate molecule from theattenuator-adaptor molecule; and (iii) deoxynucleotides that arecomplementary to the attenuator sequence of the attenuator molecule,under conditions that allow extension of the substrate polynucleotide totail the substrate; (2) ligating the adaptor sequence X to the substratepolynucleotide and dissociating the attenuator molecule from theseparate polynucleotide; (3) adding a primer complementary to a sequencein the substrate polynucleotide under conditions wherein the primerhybridizes to the substrate polynucleotide; (4) adding a polymerase anddeoxynucleotides to perform polymerase extension from the primer toproduce a second strand polynucleotide complementary to the substratepolynucleotide and create a double stranded substrate molecule; (5)ligating the adaptor molecule to the double stranded substrate molecule;(6) optionally degrading the second strand polynucleotide. In someembodiments, the primer is sufficiently complementary to a sequence inthe substrate polynucleotide to hybridize under appropriate conditionsto sequence X. In further embodiments, the primer is a target-specificprimer sufficiently complementary to hybridize under appropriateconditions to a sequence in the substrate molecule other than sequenceX. In additional embodiments, the substrate polynucleotide is a singlestrand DNA polynucleotide, and in still further embodiments thesubstrate polynucleotide is a ribonucleic acid (RNA).

A kit comprising any of the compositions disclosed herein is alsoprovided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts attenuated, polymerase-mediated poly(dA) DNA tailing inthe presence of long (>20b) complementary poly(dT) polynucleotide. TheFigure exemplifies, without limitation, using TdT enzyme for the tailingreaction. FIG. 1 discloses SEQ ID NOs 62, 62, 63 and 62, respectively,in order of appearance.

FIG. 2 depicts attenuated polymerase-mediated poly(dA) tailing withshort (12-14b) complementary poly (dT) polynucleotides. The Figureexemplifies, without limitation, using TdT enzyme for the tailingreaction. FIG. 2 discloses SEQ ID NOs 64, 65, 64, 66, 64, 67, 64, 68, 64and 64, respectively, in order of appearance.

FIG. 3 depicts the expected kinetics of poly(dA) tailing in the presenceof short poly(dT) attenuator molecule.

FIG. 4 depicts a method wherein attenuated polymerase-mediated poly(dA)tailing is performed with degradable attenuator polynucleotidecontaining dU bases. The Figure exemplifies, without limitation, usingTdT enzyme for the tailing reaction. FIG. 4 discloses SEQ ID NOs 69, 66,70 and 66, respectively, in order of appearance.

FIG. 5 depicts the attenuated polymerase-mediated poly(dT), poly(dG) andpoly(dC) tailing with a long (>20b) complementary attenuatorpolynucleotide. The Figure exemplifies, without limitation, using TdTenzyme for the tailing reaction. FIG. 5 discloses SEQ ID NOs 68, 64, 68,22, 22, 71 and 71, respectively, in order of appearance.

FIG. 6 depicts attenuated polymerase-mediated poly(dA), poly(dT),poly(dG) and poly(dC) tailing with degradable attenuatorribo-polynucleotides. The Figure exemplifies, without limitation, usingTdT enzyme for the tailing reaction. FIG. 6 discloses SEQ ID NOs 25, 64,25 and 64, respectively, in order of appearance.

FIG. 7 depicts attenuated poly (A)-polymerase-mediated poly (rA) tailingof RNA substrates using a complementary DNA poly (dT)₃₀ polynucleotide(SEQ ID NO: 62). FIG. 7 discloses SEQ ID NOs 62, 62, 73 and 62,respectively, in order of appearance.

FIG. 8 depicts attenuated poly (U)-polymerase-mediated poly (rU) tailingof RNA substrates using complementary DNA poly (dA)₃₀ polynucleotide(SEQ ID NO: 68). FIG. 8 discloses SEQ ID NOs 68, 68, 72 and 68,respectively, in order of appearance.

FIG. 9A depicts 3′-end adaptor attachment to single-stranded DNA or RNAmolecules using a limited tailing-ligation reaction.

FIG. 9B depicts 3′-end adaptor attachment to single-stranded DNA and RNAmolecules using a limited tailing-polymerase-extension reaction.

FIG. 10 depicts covalent immobilization of single-stranded DNA and RNAto a solid support using either (A) a coupled limited tailing-ligationreaction or (B) a limited tailing-polymerase extension reaction.

FIG. 11 depicts second adaptor attachment by blunt-end or dA/dTligation.

FIG. 12 depicts second adaptor attachment by a coupled limitedtailing-ligation reaction. gDNA represents genomic DNA.

FIG. 13 depicts second adaptor attachment by a limitedtailing-polymerase-extension reaction.

FIG. 14 depicts the predicted length of poly(dA) sequences introduced bya polymerase enzyme in the presence of a long poly(dT) attenuatormolecule as a function of the reaction temperature. The Figureexemplifies, without limitation, using TdT enzyme for the tailingreaction.

FIG. 15 shows an experimentally determined time course of poly(dA)tailing of single-stranded DNA template by TdT enzyme in the absence andin the presence of long degradable DNA attenuator molecule (TTTTTU)6TTT(SEQ ID NO: 43). FIG. 15 discloses SEQ ID NOs 86 and 74-76,respectively, in order of appearance.

FIGS. 16A-B show controlled poly(dA) tailing of single-stranded DNAtemplate by TdT in the presence of short attenuator molecules. FIG. 16Ashows the effect of attenuator length and reaction temperature. FIG. 16Bshows the effect of long incubation time with short attenuator. FIGS.16A-B disclose SEQ ID NOs 74-76, 86, 77, 78, 68 and 75, respectively, inorder of appearance.

FIG. 17 shows controlled and uncontrolled poly(dA) TdT tailing ofdouble-stranded DNA templates. FIG. 17 discloses SEQ ID NOs 86 and 79,respectively, in order of appearance.

FIG. 18 shows controlled poly(dA) TdT tailing of single-stranded DNAtemplates with randomized ends. FIG. 18 discloses SEQ ID NO: 86.

FIG. 19 shows controlled TdT tailing of single-stranded DNA templateswith poly (dT), poly (dC) and poly (dG) tails.

FIG. 20 shows controlled poly(rA) tailing of single-stranded RNAtemplate by E. coli poly (A) polymerase.

FIGS. 21A-C show controlled poly(rU) tailing of single-stranded RNAtemplate by the yeast (S. pombe) poly (U) polymerase. FIG. 21A shows atime course in the presence of short DNA attenuator. FIG. 21B shows theeffect of long DNA and RNA attenuators. FIG. 21C shows the effect oflong RNA oligonucleotide attenuator.

FIG. 22 shows simultaneous controlled DNA TdT tailing and ligation toattenuator-adaptor complex in solution and solid phase.

FIG. 23 shows experimental data illustrating simultaneous controlledpoly(A) polymerase tailing and ligation of single-stranded RNA templateto attenuator-adaptor complex in solution.

FIGS. 24A-C depict diagrams illustrating the process of simultaneouscontrolled tailing, ligation and immobilization of single-stranded DNAand RNA. FIG. 24A shows the process of simultaneous controlled tailing,ligation and immobilization of a single-stranded DNA withattenuator-adaptor complex in solution (DNA). FIG. 24A discloses SEQ IDNOs 2, 27, 26, 80, 27, 26, 81, 82 and 26, respectively, in order ofappearance. FIG. 24B shows the process of simultaneous controlledtailing, ligation and immobilization of a single-stranded DNA withattenuator-adaptor complex immobilized to magnetic beads (DNA). FIG. 24Bdiscloses SEQ ID NOs 2, 27, 26, 80, 27, 26, 82, 26, 27 and 26,respectively, in order of appearance. FIG. 24C shows the process ofsimultaneous controlled tailing, ligation and immobilization of asingle-stranded RNA with attenuator-adaptor complex in solution (RNA).FIG. 24C discloses SEQ ID NOs 13, 28, 29, 83, 28, 29, 87, 84 and 29,respectively, in order of appearance. FIGS. 24A-B exemplify, withoutlimitation, using TdT enzyme for the tailing reaction.

FIG. 25 depicts a method of NGS library synthesis using controlledhomopolymer tailing and ligation followed by circularization of thesubstrate polynucleotide.

FIG. 26 shows an alternative method for NGS library synthesis usingcontrolled homopolymer tailing followed by polymerase extension andcircularization.

FIG. 27 depicts a method of NGS library synthesis using controlledhomopolymer tailing and ligation followed by reverse strand synthesisand blunt adaptor ligation.

FIG. 28 shows an alternative method for NGS library constructioncomprising controlled tailing and polymerization followed by reversestrand synthesis and blunt ligation.

FIG. 29 depicts another method of NGS library preparation that comprisestwo sequential tailing and ligation reactions.

FIG. 30 is a schematic representation of the preparation of a fragmentNGS library by random primer extension and controlled tailing andligation of the extension products.

FIG. 31 is a schematic representation of the preparation of a targetedNGS library by target-specific primer extension and controlled tailingand ligation of the extension products.

FIG. 32 is a schematic representation of the preparation of a targetedNGS library using a controlled tailing and ligation reaction followed byreplication and adaptor ligation.

FIG. 33 is a schematic representation of various approaches forpreparing a targeted NGS library using a controlled tailing-adaptorligation reaction as contemplated by the disclosure.

FIG. 34 shows experimental data regarding Example 12, gel 1, which is acontrolled tailing and ligation reaction with attenuator-adaptormolecules that comprise an additional 3′ domain of random basecomposition.

FIG. 35 shows experimental data regarding Example 12, gel 2, which is acontrolled tailing and ligation reaction with attenuator-adaptormolecules that comprise an additional 3′ domain of random basecomposition.

FIG. 36 shows experimental data regarding a controlled tailing andligation reaction with dinucleotide attenuator-adaptors.

FIG. 37 depicts various methods of blunt or TA adaptor ligation as partof a whole genome or targeted library preparation.

FIG. 38 is a graph demonstrating that kinetics of poly(dA) tailing inthe presence of a long attenuator molecule (>20b).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a composition comprising a nucleic acid polymeraseand an attenuator molecule. The composition is used in a method foradding one or more nucleotides to a substrate polynucleotide in acontrolled manner, thereby adding a desired number of nucleotides to thesubstrate polynucleotide. By way of example and without limitation,elongation of a strand of a polynucleotide using a nucleic acidpolymerase is regulated by addition to the elongation reaction mixtureof an attenuator molecule that binds to a newly added tail sequencecreated by the polymerase, thereby forming a duplex structure and thusreducing the rate of the polymerization process. As a result, thereaction rate is controlled and tail sequences of a desired, limitedsize are added to substrate polynucleotides in the reaction mixture andthe tails added to the substrate polynucleotides in the reaction mixturehave a very narrow size-distribution.

The disclosure provides methods and reagents that allow the attenuationand control of addition of a tail sequence to the end of a substratepolynucleotide by nucleic acid polymerases. The disclosure also providescompositions for the reactions which are used as a basis for methods,and kits designed to carry out the methods, for size-controlled tailingof a substrate polynucleotide with addition of a tail sequence asdescribed herein using a nucleic acid polymerase. The compositions, andmethods, and kits for carrying out the methods, provide for efficientand controlled attachment of a tail sequence to the substratepolynucleotide.

The term “tailing” as used herein is interchangeable with the terms“controlled tailing” and “limited tailing.”

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

It is also noted that the term “about” as used herein is understood tomean approximately. “Destabilize,” when referring to a molecule of thedisclosure (for example and without limitation, an attenuator molecule),means to be rendered susceptible to breakage. Breakage occurs via, forexample and without limitation, incubating the molecule at hightemperature (about 80° C. or higher), incubating the molecule with anapurinic/apyrimidinic endonuclease or combinations thereof.

Nucleic Acid Polymerases

The disclosure contemplates a composition comprising an attenuatormolecule and a nucleic acid polymerase. Methods of the disclosure alsoinclude those that utilize additional nucleic acid polymerases. Anypolymerase that can add a specific homopolymeric sequence to the 3′ endof a nucleic acid is contemplated for use in the methods describedherein.

In some aspects, the nucleic acid polymerase is a DNA polymerase, and inone specific aspect the DNA polymerase is terminal deoxynucleotidyltransferase (TdT). It is also contemplated that the nucleic acidpolymerase is a RNA polymerase, and in these aspects the RNA polymeraseis selected from the group consisting of poly(A) polymerase and poly(U)polymerase. In one specific aspect the RNA polymerase is RNA-specificribonucleotidyl transferase. These polymerases all represent a family atemplate-independent polymerases.

To the extent that an enzyme can add a specific homopolymeric sequenceto the 3′ end of a nucleic acid, non-limiting examples of enzymes thatmay be used to practice the present invention include but are notlimited to terminal deoxynucleotidyl transferase (TdT), E. coli Poly(A)Polymerase, S. pombe poly(U) Polymerase and yeast poly(A) Polymerase.Addition of a repetitive sequence to the 3′ end of a substrate can beperformed by a DNA telomerase.

Other polymerases that may be used to practice the methods disclosedherein include but are not limited to Deep VentR™ DNA Polymerase,LongAmp™ Taq DNA Polymerase, Phusion™ High-Fidelity DNA Polymerase,Phusion™ Hot Start High-Fidelity DNA Polymerase, VentR® DNA Polymerase,DyNAzyme™ II Hot Start DNA Polymerase, Phire™ Hot Start DNA Polymerase,Crimson LongAmp™ Taq DNA Polymerase, DyNAzyme™ EXT DNA Polymerase,LongAmp™m Taq DNA Polymerase, Taq DNA Polymerase with Standard Taq(Mg-free) Buffer, Taq DNA Polymerase with Standard Taq Buffer, Taq DNAPolymerase with ThermoPol II (Mg-free) Buffer, Taq DNA Polymerase withThermoPol Buffer, Crimson Taq™ DNA Polymerase, Crimson Taq™ DNAPolymerase with (Mg-free) Buffer, VentR® (exo−) DNA Polymerase, HemoKlenTaq™, Deep VentR™ (exo−) DNA Polymerase, ProtoScript® AMV FirstStrand cDNA Synthesis Kit, ProtoScript® M-MuLV First Strand cDNASynthesis Kit, Bst DNA Polymerase, Full Length, Bst DNA Polymerase,Large Fragment, Taq DNA Polymerase with ThermoPol Buffer, 9° Nm DNAPolymerase, Crimson Taq™ DNA Polymerase, Crimson Taq™ DNA Polymerasewith (Mg-free) Buffer, Deep VentR™ (exo−) DNA Polymerase, Deep VentR™DNA Polymerase, DyNAzyme™ EXT DNA Polymerase, DyNAzyme™ II Hot Start DNAPolymerase, Hemo KlenTaq™, Phusion™ High-Fidelity DNA Polymerase,Phusion™ Hot Start High-Fidelity DNA Polymerase, Sulfolobus DNAPolymerase IV, Therminator™ y DNA Polymerase, Therminator™ DNAPolymerase, Therminator™ II DNA Polymerase, Therminator™ III DNAPolymerase, VentR® DNA Polymerase, VentR® (exo−) DNA Polymerase, Bst DNAPolymerase, Large Fragment, DNA Polymerase I (E. coli), DNA PolymeraseI, Large (Klenow) Fragment, Klenow Fragment (3′->5-exo−), phi29 DNAPolymerase, T4 DNA Polymerase, T7 DNA Polymerase (unmodified), ReverseTranscriptases and RNA Polymerases, AMV Reverse Transcriptase, M-MuLVReverse Transcriptase, phi6 RNA Polymerase (RdRP), SP6 RNA Polymerase,and T7 RNA Polymerase.

Ligases that may be used to practice the methods of the disclosureinclude but are not limited to T4 DNA ligase, T4 RNA ligase, E. coli DNAligase and E. coli RNA ligase.

Attenuator Molecule

The present disclosure provides compositions and methods that comprisean attenuator molecule (used interchangeably herein with “attenuatorpolynucleotide”). The attenuator is, in various aspects, apolynucleotide, an immobilized molecule, a polypeptide, apolysaccharide, a linear molecule, a circular molecule, a singlestranded molecule, a partially double stranded molecule, a peptidenucleic acid, a Schizophyllan polysaccharide, a locked nucleic acidand/or combinations thereof.

The number of nucleotides added to a substrate polynucleotide isdependent on the conditions under which the reaction is performed. Insome aspects, an attenuator molecule is a polynucleotide that hybridizesto a sequence added to a substrate polynucleotide with polymeraseactivity in the composition, wherein the number of nucleotides added tothe substrate polynucleotide is essentially equal to the number ofnucleotides in the attenuator molecule with which the tail sequence canassociate. In some aspects, the number of nucleotides added to thesubstrate polynucleotide is essentially equal to a multiple of thenumber of nucleotides in the attenuator molecule with which the tailsequence can associate. By way of example and without limitation, if thelength of an attenuator molecule is 13 nucleotides, then the number ofnucleotides that are added to the substrate polynucleotide isessentially 13 nucleotides. Depending on the conditions under which thereaction is performed, however, the number of nucleotides that are addedto the substrate polynucleotide is a multiple of 13, or essentially 26(two times the length of the attenuator molecule), or essentially 39(three times the length of the attenuator molecule), or essentially 52(four times the length of the attenuator molecule) or more multiples ofthe length of the attenuator molecule. In some aspects, therefore, thetail sequence of the substrate polynucleotide interacts with more thanone attenuator molecule. In some aspects, the number of nucleotidesadded to a tail of the substrate polynucleotide is less than the lengthof the attenuator molecule.

In some aspects, the number of nucleotides added to the substratepolynucleotide is determined not by the length of attenuator but by thetemperature and/or salt concentration at which the reaction isperformed. In some aspects, a higher temperature and a lower saltconcentration will result in more nucleotides being added to the tail ofthe substrate polynucleotide. It is contemplated that the number ofnucleotides added to the tail of the substrate polynucleotide willincrease until a certain number is reached, the number being determinedby the conditions (i.e., temperature and/or salt concentration) at whicha stable duplex is formed between the substrate polynucleotide and theattenuator molecule. Formation of a stable duplex with the attenuatormolecule inhibits further addition of nucleotides to the tail of thesubstrate polynucleotide, and thus it is the T_(m) of the stable duplexthat dictates the number of nucleotides that are added to the tail ofthe substrate polynucleotide.

In further embodiments, an attenuator molecule further comprises anadaptor sequence as described herein below. In aspects wherein theattenuator molecule is a polynucleotide, it is contemplated that thehomopolymeric portion of the polynucleotide is the “attenuator” portion.In aspects wherein the polynucleotide comprises nucleotides in additionto the homopolymeric sequence, the polynucleotide is referred to hereinas an “attenuator-adaptor” molecule. The additional nucleotides can bepart of the same polynucleotide, or can be present in two separatepolynucleotides that are hybridized to each other. Thus, in stillfurther embodiments, the attenuator molecule and the adaptor molecule(which comprises the adaptor sequence) are two separate polynucleotidesthat are at least partially hybridized together. In various embodiments,a single polynucleotide comprises an attenuator portion and an adaptorsequence. In various aspects, the polynucleotide forms a hairpinstructure to create a partially double stranded polynucleotide. In thishairpin configuration, the attenuator portion is single-stranded, andthe adaptor sequence is double-stranded.

In additional embodiments, the attenuator or attenuator-adaptor moleculecomprises a dinucleotide polymer sequence instead of a homopolymersequence. Thus, in various embodiments, the disclosure contemplates thatthe dinucleotide portion of the attenuator comprises a plurality ofrandom sequences comprised of the following dinucleotide combinations:(i) dG or dC; (ii) dA or dT; (iii) dG or dT; (iv) dG or dA; (v) dA ordC; or (vi) dC or dT. The dinucleotide sequences, in variousembodiments, comprise mixtures of ribonucleotides anddeoxyribonucleotides. In these embodiments, it is further contemplatedthat the nucleotide mix used for the tailing reactions comprise thecomplementary nucleotides to those used in the homopolymeric portion ofthe attenuator. During the tailing process, a plurality of random tailsequences comprised of the dinucleotides complementary to thedinucleotide attenuator are generated. In the disclosure, variousembodiments are described with reference to a homopolymer sequence or ahomopolymer or dinucleotide sequence. The worker or skill in the artwill appreciate, however, that in instances wherein only a homopolymeris described, the method can readily be carried out using a dinucleotidesequence with modifications as described herein.

In still further embodiments, the attenuator or attenuator-adaptormolecule further comprises an additional sequence on its 3′ end, whereinthe additional sequence is not a homopolymer or a dinucleotide sequencebut comprises a random nucleotide sequence which in various aspects,comprises ribonucleotides, deoxyribonucleotides or a combinationthereof. The additional random sequence is from about 1 to about 50nucleotides or more in length, or from about 1 to about 5 nucleotides,or from about 1 to about 10, 20, 30, 40 or 50 nucleotides, or from about5 to about 10 nucleotides, or from about 4 to about 7 nucleotides, orfrom about 5 to about 15 nucleotides, or from about 10 to about 15nucleotides, or from about 10 to about 20, 30, 40 or 50 nucleotides inlength, or from about 5 to about 10, 20, 30, 40 or 50 nucleotides, orfrom about 10 to about 20, 30, 40 or 50 nucleotides in length, or fromabout 20 to about 30, 40 or 50 nucleotides in length. In furtherembodiments, the additional sequence is about 1, about 2, about 3, about4, about 5 about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50 or more nucleotides in length. In stillfurther embodiments, the additional sequence is at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 7, at least about 8, at least about 9, atleast about 10, at least about 11, at least about 12, at least about 13,at least about 14, at least about 15, at least about 16, at least about17, at least about 18, at least about 19, at least about 20, at leastabout 21, at least about 22, at least about 23, at least about 24, atleast about 25, at least about 26, at least about 27, at least about 28,at least about 29, at least about 30, at least about 31, at least about32, at least about 33, at least about 34, at least about 35, at leastabout 36, at least about 37, at least about 38, at least about 39, atleast about 40, at least about 41, at least about 42, at least about 43,at least about 44, at least about 45, at least about 46, at least about47, at least about 48, at least about 49, at least about 50 or morenucleotides in length. In further embodiments, the additional sequenceis 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides inlength.

It will be understood that not all of the attenuator orattenuator-adaptor molecules used in a given reaction are uniform insize. Thus, in various embodiments, the homopolymer portion and theadditional sequence portion of the attenuator molecule are each fromabout 1 to about 500 nucleotides in length. In further embodiments, thedisclosure contemplates that an attenuator molecule that is apolynucleotide comprises a homopolymer portion and an additionalsequence portion, each of which is at least 1 nucleotide and up to about5, 10, 20, 30, 50, 100, 200, 300 or 500 nucleotides, at least 2nucleotides and up to about 5, 10, 20, 30, 50, 100, 200, 300 or 500nucleotides, at least 5 nucleotides and up to about 10, 20, 30, 50, 100,200, 300 or 500 nucleotides, at least 5 nucleotides and up to about 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides, at least 10 and up toabout 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides, at least 10 and upto about 20, 30, 40, 50, 100, 200, 300 or 500 nucleotides, at least 20and up to about 30, 40, 50, 100, 200, 300 or 500 nucleotides, at least50 and up to about 70, 100, 200, 300 or 500 nucleotides or at least 50and up to about 100, 300, 400 or 500 nucleotides.

It is contemplated that “hybridization” as used herein encompasses anyassociation between the attenuator molecule and the tail sequence of thesubstrate polynucleotide. For example and without limitation, theassociation can be the result of Watson-Crick base-pairing, or othertypes of base-pairing between the attenuator molecule and the substratepolynucleotide such as DNA, RNA and peptide nucleic acids (PNA).

In some embodiments, the attenuator molecule is a polynucleotide and ithybridizes to the tail portion of the substrate polynucleotide understringent conditions. “Stringent conditions” as used herein can bedetermined empirically by the worker of ordinary skill in the art andwill vary based on, for example and without limitation, the length ofthe attenuator molecule and the tail sequence of the substratepolynucleotide, concentrations of the attenuator molecule and thesubstrate polynucleotide, the salt concentration (i.e., ionic strength)in the hybridization buffer, the temperature at which the hybridizationis carried out, length of time that hybridization is carried out, andpresence of factors that affect surface charge of the attenuatormolecule and the tail sequence of the substrate polynucleotide. Ingeneral, stringent conditions are those in which the tail sequence ofthe substrate polynucleotide is able to bind to its complementarysequence preferentially and with higher affinity relative to any otherregion on the attenuator molecule. Exemplary stringent conditions forhybridization to its complement of a tail sequence of a substratepolynucleotide sequence having 20 bases include without limitation about50 mM salt (Na±), and an annealing temperature of about 60° C. For alonger sequence, specific hybridization is achieved at highertemperature. In general, stringent conditions are such that annealing iscarried out about 5° C. below the melting temperature of the substratepolynucleotide. The “melting temperature” is the temperature at which50% of attenuator molecules that are complementary to a substratepolynucleotide in equilibrium at definite ion strength, pH andconcentration, dissociate from the substrate polynucleotide. Asdescribed further herein below, the temperature at which thehybridization and extension is performed is, in various aspects, relatedto the addition of nucleotides to the substrate polynucleotide.

In certain embodiments where the attenuator molecule is apolynucleotide, the attenuator polynucleotide is single stranded or atleast partially double stranded inasmuch as the double strandedpolynucleotide is able to associate with the tail sequence added to thesubstrate polynucleotide. In further embodiments, the attenuatormolecule is a circular molecule comprising a homopolymeric nucleotidesequence that is able to associate with the tail sequence added to thesubstrate polynucleotide.

In further embodiments, the attenuator molecule that is a polynucleotidecomprises a nucleotide selected from the group consisting ofT-deoxythymidine 5′-monophosphate (dTMP), 2′-deoxyguanosine5′-monophosphate (dGMP), 2′-deoxyadenosine 5′-monophosphate (dAMP),2′-deoxycytidine 5′-monophosphate (dCMP), 2′-deoxyuridine5′-monophosphate (dUMP), thymidine monophosphate (TMP), guanosinemonophosphate (GMP), adenosine monophosphate (AMP), cytidinemonophosphate (CMP), uridine monophosphate (UMP), a base analog, andcombinations thereof. It is also contemplated that the attenuatormolecule polynucleotide comprises a modified nucleotide as definedherein.

In related aspects, the attenuator molecule comprises a homopolymericmolecule such as poly 2′-deoxyadenosine 5′-monophosphate (dAMP) (polydA), poly T-deoxythymidine 5′-monophosphate (dTMP) (poly dT), poly2′-deoxycytidine 5′-monophosphate (poly dC), poly 2′-deoxyguanosine5′-monophosphate (poly dG), poly 2′-deoxyuridine 5′-monophosphate (polydU), poly adenosine monophosphate (poly rA), poly uridine monophosphate(poly U), poly cytidine monophosphate (poly rC), poly guanosinemonophosphate (poly rG) or a heteropolymeric molecule comprisingcombinations of dA and rA bases, or dT, dU and U bases, or dC and rCbases, or dG and rG bases.

In various aspects, the attenuator molecule comprises 1, 5, 10, 20, 30,50, 100 or more nucleotides. Indeed, the disclosure contemplates that anattenuator molecule that is a polynucleotide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000 or more nucleotides. In furtherembodiments, the disclosure contemplates that an attenuator moleculethat is a polynucleotide comprises at least 1 nucleotide and up to about5, 10, 20, 50, 100, 200, 500 or 1000 nucleotides, at least 2 nucleotidesand up to about 5, 10, 20, 50, 100, 200, 500 or 1000 nucleotides, atleast 5 nucleotides and up to about 10, 20, 50, 100, 200, 500 or 1000nucleotides, at least 5 nucleotides and up to about 10, 15, 20, 25, 30,35, 40, 45 or 50 nucleotides, at least 10 and up to about 15, 20, 25,30, 35, 40, 45 or 50 nucleotides, at least 10 and up to about 20, 30,40, 50, 100, 200, 500 or 1000 nucleotides, at least 20 and up to about30, 40, 50, 100, 200, 500 or 1000 nucleotides, at least 50 and up toabout 70, 100, 200, 500, 700 or 1000 nucleotides or at least 50 and upto about 100, 500, 750, 800 or 1000 nucleotides.

In various aspects the attenuator molecule comprises a blocking group. Ablocking group as used herein is a moiety that prevents extension by anenzyme that is capable of synthesizing a polynucleotide by addition ofnucleotides. Blocking groups include but are not limited to a phosphategroup, a dideoxynucleotide, a ribonucleotide (in aspects wherein a TdTenzyme is used), deoxynucleotides (in aspects wherein a poly(A) and/or apoly(U) polymerase is used), an amino group, a three or six carbonglycol spacer (and in one aspect the six carbon glycol spacer ishexanediol) and an inverted deoxythymidine (dT).

In another aspect of the disclosure, the attenuator molecule isdegradable. The degradable attenuator molecule comprises, in variousaspects, dU bases and degradation is caused by contact with adU-glycosylase followed by incubation at a temperature that is above 80°C., or by contact with a mixture of a dU-glycosylase and anapurinic/apyrimidinic endonuclease.

It is also contemplated that the attenuator molecule comprises, in someembodiments, ribonucleotides and has a sequence that is degradable witha ribonuclease. In various aspects, the ribonuclease is selected fromthe group consisting of RNase H, RNase HII, RNase A, and RNase T1 underconditions sufficient for ribonuclease activity. In a related aspect,the attenuator molecule comprises deoxyribonucleotides and has asequence that is degradable with a DNA-specific nuclease. TheDNA-specific nuclease is, in some aspects, DNase I.

The attenuator molecule, in further embodiments, further comprises anadaptor sequence, an identifier tag sequence, or both. An “adaptorsequence” provides a priming sequence for both amplification andsequencing of nucleic acid fragments and is used, in some aspects, fornext generation sequencing applications. In further aspects, an “adaptorsequence” is used as a promoter sequence for generation of RNAmolecules, wherein the promoter sequence is, for example and withoutlimitation, a T7 promoter sequence or an SP6 promoter sequence. Any RNApromoter that is known in the art is contemplated as an adaptorsequence.

In some embodiments, the “identifier tag sequence” is a sequence thatuniquely identifies a particular substrate or attenuator molecule. Inone aspect, the identifier tag sequence is a barcode.

In some aspects the attenuator molecular is a polypeptide. As usedherein, the term “polypeptide” refers to peptides, proteins, polymers ofamino acids and antibodies that are naturally derived, syntheticallyproduced, or recombinantly produced. Polypeptides also includelipoproteins and post-translationally modified proteins, such as, forexample, glycosylated proteins, as well as proteins or proteinsubstances that have D-amino acids, modified, derivatized, ornon-naturally occurring amino acids in the D- or L-configuration and/orpeptomimetic units as part of their structure.

With regard to proteins, attenuator molecules contemplated include fulllength protein and fragments thereof which retain the desired propertyof the full length proteins. Fusion proteins, including fusion proteinswherein one fusion component is a fragment or a mimetic, are alsocontemplated.

Antibody attenuator molecules include fragments and derivatives of fulllength antibodies. Specifically contemplated fragments and derivativesinclude, but are not limited to, Fab′ fragments, F(ab)₂ fragments, Fvfragments, Fc fragments, one or more complementarity determining regions(CDR) fragments, individual heavy chains, individual light chain,dimeric heavy and light chains (as opposed to heterotetrameric heavy andlight chains found in an intact antibody, single chain antibodies(scAb), humanized antibodies (as well as antibodies modified in themanner of humanized antibodies but with the resulting antibody moreclosely resembling an antibody in a non-human species), chelatingrecombinant antibodies (CRABs), bispecific antibodies and multispecificantibodies, and other antibody derivative or fragments known in the art.

DNA and RNA binding proteins are contemplated for use in the methods andcompositions of the disclosure. DNA-binding proteins are proteins thatare comprised of DNA-binding domains and thus have a specific or generalaffinity for single stranded DNA [Travers, DNA-protein Interactions.Springer, 1993; Pabo et al., Protein-DNA recognition. Annu Rev Biochem.53: 293-321 (1984)]. Polypeptides that bind to homopolymeric sequencesare known in the art [Lobanenkov et al., Eur J Biochem. 159(1): 181-8(1986); Travers, Annu Rev Biochem, 58: 427-452 (1989); Ostrowski et al.,Proc. Natl. Acad. Sci. (USA) 98(16): 9044-9049 (2001)], and contemplatedfor use herein.

RNA-binding proteins are typically cytoplasmic and nuclear proteins thatassociate with double strand or single strand RNAs through an RNArecognition motif (RRM). RNA-binding proteins may regulate thetranslation of RNA, and post-transcriptional events such as, withoutlimitation, RNA splicing and editing. Some examples of RNA bindingproteins include, without limitation, translation initiation factorsthat bind RNA, polyA-binding proteins, snRNPs, and double strandedRNA-specific adenosine deaminase (ADAR).

Another type of attenuator molecule contemplated by the disclosure ispolysaccharide Schizophyllan that can form non-Watson-Crick typemacromolecular complexes with poly(C), poly(A), poly(dA) and poly(dT)homo-polymers. Schizophyllan (SPG) is a natural 13-(1,3)-D¬glucanexisting as a triple helix in water and as a single chain indimethylsulfoxide (DMSO), respectively [Matsumoto et al., BiochimBiophys Acta. 1670(2): 91-104 (2004)]. Schizophyllan has glucose sidechain through a β-1,6-glycosil bond. It has been shown thatSchizophyllan can form a complex with single stranded polynucleotides.In the presence of polynucleotides, single chain SPG in an aqueoussolution forms a triple stranded complex that consist of two SPG chainsand a polynucleotide chain. Schizophyllan can form a triple strandedcomplex with a single stranded polynucleotide through hydrogen bondingand hydrophobic interaction. It was shown that the polynucleotide wasprotected from nuclease attack in forming the complex withSchizophyllan, and Schizophyllan enhanced antisense efficiency [Sakuraiet al., Nucleic Acids Research Supplement No. 1: 223-224 (2001)].

Regardless of the type of attenuator molecule, it is contemplated thatin some aspects the attenuator molecule is immobilized on a support asdescribed herein below.

Substrate Polynucleotide

A substrate polynucleotide is a polynucleotide, modified polynucleotideor combination thereof as described herein below. The substratepolynucleotide is, in various embodiments, DNA, RNA, or a combinationthereof. The substrate polynucleotide to which the tail is added iseither single stranded or double stranded. In further aspects, thesubstrate polynucleotide can be a triple helix, a G-quartet, or othermulti-strand structure. In another embodiment, the substratepolynucleotide is chemically treated nucleic acid, including but notlimited to embodiments wherein the substrate polynucleotide isbisulfite-treated DNA to detect methylation status by NGS.

It is contemplated that substrate polynucleotides are obtained fromnaturally occurring sources or they can be synthetic. The naturallyoccurring sources are RNA and/or genomic DNA from a prokaryote or aeukaryote. For example and without limitation, the source can be ahuman, mouse, virus, plant or bacteria. In various aspects, thesubstrate polynucleotide is tailed for use in assays involvingmicroarrays and creating libraries for next generation nucleic acidsequencing. Tailed substrate polynucleotides can also be used forefficient cloning of DNA and RNA.

If the source of the substrate polynucleotide is genomic DNA, it iscontemplated that in some embodiments the genomic DNA is fragmentedprior to its being tailed. Fragmenting of genomic DNA is a generalprocedure known to those of skill in the art and is performed, forexample and without limitation in vitro by shearing (nebulizing) theDNA, cleaving the DNA with an endonuclease, sonicating the DNA, byheating the DNA, by irradiation of DNA using alpha, beta, gamma or otherradioactive sources, by light, by chemical cleavage of DNA in thepresence of metal ions, by radical cleavage and combinations thereof.Fragmenting of genomic DNA can also occur in vivo, for example andwithout limitation due to apoptosis, radiation and/or exposure toasbestos. According to the methods provided herein, a population ofsubstrate polynucleotides are not required to be of a uniform size.Thus, the methods of the disclosure are effective for use with apopulation of differently-sized substrate polynucleotide fragments.

The substrate polynucleotide, as disclosed herein, is either singlestranded or double stranded and comprises a 3′ overhang. In some aspectsthe substrate polynucleotide is double stranded and comprises a bluntend. In other aspects, the double stranded substrate polynucleotidecomprises a 3′ recessed end. In all aspects, the substratepolynucleotide comprises a free 3′ hydroxyl group. The length of anoverhang or recessed end of a substrate polynucleotide can be varied. Invarious aspects, the length of an overhang or recessed end of asubstrate polynucleotide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morenucleotides in length. In specific aspects, a 3′ overhang that is 3nucleotides in length is a more efficient substrate polynucleotide thana 3′ overhang that is either 2 nucleotides in length or 1 nucleotide inlength. A population of substrate polynucleotides in various aspects,includes those wherein more than one of the above-mentioned types ofsubstrate polynucleotides are present in a single reaction.

In some embodiments, it is contemplated that the substratepolynucleotide is immobilized on a solid surface as described hereinbelow. Immobilization of the substrate polynucleotide results, in oneaspect, from its ligation to an attenuator-adaptor molecule as describedbelow.

The length of a substrate polynucleotide is contemplated to be betweenabout 3 and about 1×10⁶ nucleotides. In some aspects, the length of thesubstrate polynucleotide is between about 10 and about 3000 nucleotides,or between about 40 and about 2000 nucleotides, or between about 50 andabout 1000 nucleotides, or between about 100 and about 500 nucleotides,or between about 1000 and about 5000 nucleotides, or between about10,000 and 50,000 nucleotides, or between about 100,000 and 1×10⁶nucleotides. In further aspects, the length of the substratepolynucleotide is at least 3 and up to about 50, 100 or 1000nucleotides; or at least 10 and up to about 50, 100 or 1000 nucleotides;or at least 100 and up to about 1000, 5000 or 10000 nucleotides; or atleast 1000 and up to about 10000, 20000 and 50000; or at least 10000 andup to about 20000, 50000 and 100,000 nucleotides; or at least 20000 andup to about 100,000, 200,000 or 500,000 nucleotides; or at least 200,000and up to about 500,000, 700,000 or 1,000,000 nucleotides. In variousaspects, the length of the substrate polynucleotide is about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, about 100, about 110, about 120, about 130, about 140, about150, about 160, about 170, about 180, about 190, about 200, about 210,about 220, about 230, about 240, about 250, about 260, about 270, about280, about 290, about 300, about 310, about 320, about 330, about 340,about 350, about 360, about 370, about 380, about 390, about 400, about410, about 420, about 430, about 440, about 450, about 460, about 470,about 480, about 490, about 500, about 510, about 520, about 530, about540, about 550, about 560, about 570, about 580, about 590, about 600,about 610, about 620, about 630, about 640, about 650, about 660, about670, about 680, about 690, about 700, about 710, about 720, about 730,about 740, about 750, about 760, about 770, about 780, about 790, about800, about 810, about 820, about 830, about 840, about 850, about 860,about 870, about 880, about 890, about 900, about 910, about 920, about930, about 940, about 950, about 960, about 970, about 980, about 990,about 1000, about 1100, about 1200, about 1300, about 1400, about 1500,about 1600, about 1700, about 1800, about 1900, about 2000, about 2100,about 2200, about 2300, about 2400, about 2500, about 2600, about 2700,about 2800, about 2900, about 3000, about 3100, about 3200, about 3300,about 3400, about 3500, about 3600, about 3700, about 3800, about 3900,about 4000, about 4100, about 4200, about 4300, about 4400, about 4500,about 4600, about 4700, about 4800, about 4900, about 5000, 10,000,15,000, 20,000, 50,000, 100,000, 150,000, 200,000, 250,000, 300,000,350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000,750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000 or morenucleotides.

Polynucleotides

The terms “polynucleotide” and “nucleotide” or plural forms as usedherein are interchangeable with modified forms as discussed herein andotherwise known in the art. Polynucleotides as described herein refer toeither an attenuator polynucleotide or a substrate polynucleotide andcomprise, in various embodiments, a deoxyribonucleotide, aribonucleotide or a combination thereof. In further embodiments, anattenuator polynucleotide that comprises a ribonucleotide, adeoxyribonucleotide, or a combination thereof, is used in combinationwith a substrate polynucleotide that is either DNA or RNA.

In certain instances, the art uses the term “nucleobase” which embracesnaturally-occurring nucleotides as well as modifications of nucleotidesthat can be polymerized. Thus, nucleotide or nucleobase means thenaturally occurring nucleobases adenine (A), guanine (G), cytosine (C),thymine (T) and uracil (U) as well as non-naturally occurringnucleobases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine,7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin,N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC),5-(C3-C6)-alkynyl¬cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-tr¬iazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” also includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof. Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which are hereby incorporated by reference intheir entirety). In various aspects, polynucleotides also include one ormore “nucleosidic bases” or “base units” which include compounds such asheterocyclic compounds that can serve like nucleobases, includingcertain “universal bases” that are not nucleosidic bases in the mostclassical sense but serve as nucleosidic bases. Universal bases include3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole),and optionally substituted hypoxanthine. Other desirable universal basesinclude, pyrrole, diazole or triazole derivatives, including thoseuniversal bases known in the art.

Polynucleotides may also include modified nucleobases. A “modified base”is understood in the art to be one that can pair with a natural base(e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or canpair with a non-naturally occurring base. Exemplary modified bases aredescribed in EP 1 072 679 and WO 97/12896, the disclosures of which areincorporated herein by reference. Modified nucleobases include withoutlimitation, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified bases include tricyclic pyrimidinessuch as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deazaadenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Additional nucleobases include thosedisclosed in U.S. Pat. No. 3,687,808, those disclosed in The ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289¬302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity and include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

Methods of making polynucleotides of a predetermined sequence arewell-known. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides andAnalogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for both polyribonucleotidesand polydeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Polyribonucleotides can also beprepared enzymatically. Non-naturally occurring nucleobases can beincorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No.7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J.Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949(1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am.Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc.,124:13684-13685 (2002).

Modified Polynucleotides

Modified polynucleotides are contemplated for use in an attenuatormolecule or in a substrate polynucleotide wherein both one or more sugarand/or one or more internucleotide linkage of the nucleotide units inthe polynucleotide is replaced with “non-naturally occurring” groups. Inone aspect, this embodiment contemplates a peptide nucleic acid (PNA).In PNA compounds, the sugar-backbone of a polynucleotide is replacedwith an amide containing backbone. See, for example U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262, and Nielsen et al., Science, 1991,254, 1497-1500, the disclosures of which are herein incorporated byreference.

Other linkages between nucleotides and unnatural nucleotidescontemplated for the disclosed polynucleotides include those describedin U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; and 5,700,920; U.S. Patent PublicationNo. 20040219565; International Patent Publication Nos. WO 98/39352 andWO 99/14226; Mesmaeker et. al., Current Opinion in Structural Biology5:343-355 (1995) and Susan M. Freier and Karl-Heinz Altmann, NucleicAcids Research, 25:4429-4443 (1997), the disclosures of which areincorporated herein by reference.

Specific examples of polynucleotides include those containing modifiedbackbones or non-natural internucleoside linkages. Polynucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified polynucleotides that do not have a phosphorus atom intheir internucleoside backbone are considered to be within the meaningof “polynucleotide.”

Modified polynucleotide backbones containing a phosphorus atom include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Also contemplated are polynucleotides having inverted polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage, i.e. a single inverted nucleoside residue which may be abasic(the nucleotide is missing or has a hydroxyl group in place thereof).Salts, mixed salts and free acid forms are also contemplated.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, thedisclosures of which are incorporated by reference herein.

Modified polynucleotide backbones that do not include a phosphorus atomhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. In still otherembodiments, polynucleotides are provided with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and including—CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— described in U.S. Pat.Nos. 5,489,677, and 5,602,240. See, for example, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, thedisclosures of which are incorporated herein by reference in theirentireties.

In various forms, the linkage between two successive monomers in thepolynucleotide consists of 2 to 4, desirably 3, groups/atoms selectedfrom —CH₂—, —O—, —S—, —NRH—, >C═O, >C═NRH, >C═S, —Si(R″)₂—, —SO—,—S(O)₂—, —P(O)₂—, —PO(BH₃)—, —P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—,and —PO(NHRH)—, where RH is selected from hydrogen and C1-4-alkyl, andR″ is selected from C1-6-alkyl and phenyl. Illustrative examples of suchlinkages are —CH₂—CH₂—CH2-, —CO—CH₂—CHOH—CH₂—, —O—CH₂—O—, —O—CH₂—CH₂—CH═(including R5 when used as a linkage to a succeeding monomer),—CH₂—CH₂—O—, —NRH—CH₂—CH₂—, —CH₂—CH₂—NRH—, —CH₂—NRH—CH₂—O—CH₂—CH₂—NRH—,—NRH—CO—O—, —NRH—CO—NRH—, —NRH—CS—NRH—, —NRH—C(═NRH)—NRH—,—NRH—CO—CH₂—NRH—O—CO—O—, —O—CO—CH2-O—, —O—CH₂—CO—O—, —CH₂—CO—NRH—,—O—CO—NRH—, —NRH—CO—CH₂—, —O—CH₂—CO—NRH—, —O—CH₂—CH₂—NRH—, —CH═N—O—,—CH2-NRH—O—, —CH₂—O—N═ (including R5 when used as a linkage to asucceeding monomer), —CH₂—O—NRH—, —CO—NRH—CH₂—, —CH₂—NRH—O—,—CH₂—NRH—CO—, —O—NRH—CH₂—, —C—NRH, —O—CH₂—S—, —S—CH₂—O—, —CH₂—CH₂—S—,—O—CH₂ CH₂—S—, —S—CH₂—CH═ (including R5 when used as a linkage to asucceeding monomer), —S—CH₂—CH₂—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—,—CH₂—S—CH₂—, CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—, —O—SO—O—, —O—S(O)₂—O—,—OS(O)2-CH₂—, —O—S(O)₂—NRH—, —NRH—S(O)₂—CH₂—; —O—S(O)₂—CH₂—, O—P(O)₂—O—,—O—P(O,S)—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—,—S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S—, —O—PO(R″)—O—, —O—PO(OCH₂)—O—,—O—P O (O CH₂CH₃)—O—, —O—P O(O CH₂ CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHRN)—O—, —O—P(O)₂—NRH H—, —NRH—P(O)₂—O—, —O—P(O,NRH)—O—,—CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—; among whichCH₂—CO—NRH—, —CH₂—NRH—O—, —S—CH2-O—, —O—P(O)₂—O—O—P(—O,S)—O—, —NRHP(O)₂—O—, —O—P(O,NRH)—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and—O—PO(NHRN)—O—, where RH is selected form hydrogen and C1-4-alkyl, andR″ is selected from C1-6-alkyl and phenyl, are contemplated. Furtherillustrative examples are given in Mesmaeker et. al., 1995, CurrentOpinion in Structural Biology, 5: 343-355 and Susan M. Freier andKarl-Heinz Altmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

Still other modified forms of polynucleotides are described in detail inU.S. Patent Application No. 20040219565, the disclosure of which isincorporated by reference herein in its entirety.

Modified polynucleotides may also contain one or more substituted sugarmoieties. In certain aspects, polynucleotides comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Other embodiments include[(OCH₂)_(n)O]_(m),CH₃, O(CH2)_(n), OCH₃, O(CH₂)_(n),NH₂, O(CH₂)_(n).CH₃,O(CH₂)nONH₂, and O(CH₂)nON[(CH₂)nCH₃]₂, where n and m are from 1 toabout 10. Other polynucleotides comprise one of the following at the 2′position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of a polynucleotide, or a groupfor improving the pharmacodynamic properties of a polynucleotide, andother substituents having similar properties. In one aspect, amodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., 1995, HeIv. Chim. Acta,78: 486-504) i.e., an alkoxyalkoxy group. Other modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2 group, also known as2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-0-C11)-0-CH2-N(CH3)2.

Still other modifications include 2′-methoxy (2′-0-CH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2-CH═CH2), 2′-O-allyl(2′-0-CH2-CH═CH2) and 2′-fluor^(o) (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. In one aspect, a2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the polynucleotide, for example, at the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedpolynucleotides and the 5′ position of 5′ terminal nucleotide.Polynucleotides may also have sugar mimetics such as cyclobutyl moietiesin place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792,747; and 5,700,920, the disclosures of which areincorporated by reference in their entireties herein.

Further modifications include those that extend the genetic code suchas, without limitation, Iso-dC and Iso-dG. Iso-dC and Iso-dG arechemical variants of cytosine and guanine, respectively. Iso-dC willhydrogen bond with Iso-dG but not with dG. Similarly, Iso-dG will basepair with Iso-dC but not with dC [Switzer et al., Biochemistry32:10489-96 (1993)]. In these aspects, controlled tailing by addition ofIso-dC bases is achieved by using a poly (iso-dG) attenuator moleculeand vice versa.

In one aspect, a modification of the sugar includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring, thereby forming a bicyclic sugar moiety. Thelinkage is in certain aspects a methylene (—CH2-)n group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226, thedisclosures of which are incorporated herein by reference.

Labels

In some aspects of the disclosure, any polynucleotide used in themethods or compositions described herein comprises a label. In some ofthese aspects the label is fluorescent. Methods of labelingpolynucleotides with fluorescent molecules and measuring fluorescenceare well known in the art. Fluorescent labels useful in the practice ofthe invention include but are not limited to 1,8-ANS(1-Anilinonaphthalene-8-sulfonic acid), 1-Anilinonaphthalene-8-sulfonicacid (1,8-ANS), 5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0,5-FAM pH 9.0, 5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt),5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE,6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyrhodamine 6G pH7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SEpH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin,7-Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430,Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugatepH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, Alexa Fluor 488hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH7.2, Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor647 antibody conjugate pH 7.2, Alexa Fluor 647 R-phycoerythrinstreptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, AlexaFluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugatepH 7.2, Allophycocyanin pH 7.5, AMCA conjugate, Amino Coumarin, APC(allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (BlueFluorescent Protein), BO-PRO-1-DNA, BO-PRO-3-DNA, BOBO-1-DNA,BOBO-3-DNA, BODIPY 650/665-X, Me0H, BODIPY FL conjugate, BODIPY FL,Me0H, Bodipy R6G SE, BODIPY R6G, Me0H, BODIPY TMR-X antibody conjugatepH 7.2, Bodipy TMR-X conjugate, BODIPY TMR-X, Me0H, BODIPY TMR-X, SE,BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, Me0H, BODIPY TR-X, SE,BOPRO-1, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, CalciumCrimson Ca2+, Calcium Green, Calcium Green-1 Ca2+, Calcium Orange,Calcium Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue,Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibodyconjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5,CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, Cy 5.5,CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, Me0H, DAPI,DAPI-DNA, Dapoxyl (2-aminoethyl) sulfonamide, DDAO pH 9.0, Di-8 ANEPPS,Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed,DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (EnhancedGreen Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0,Erythrosin-5-isothiocyanate pH 9.0, Ethidium Bromide, Ethidiumhomodimer, Ethidium homodimer-1-DNA, eYFP (Enhanced Yellow FluorescentProtein), FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3,Fluo-3 Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1 M NaOH,Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0,Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura Red, low Ca,Fura-2 Ca2+, Fura-2, high Ca, Fura-2, no Ca, GFP (S65T), HcRed, Hoechst33258, Hoechst 33258-DNA, Hoechst 33342, Indo-1 Ca2+, Indo-1, Ca free,Indo-1, Ca saturated, JC-1, JC-1 pH 8.2, Lissamine rhodamine,LOLO-1-DNA, Lucifer Yellow, CH, LysoSensor Blue, LysoSensor Blue pH 5.0,LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0,LysoSensor Yellow pH 9.0, LysoTracker Blue, LysoTracker Green,LysoTracker Red, Magnesium Green, Magnesium Green Mg2+, MagnesiumOrange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green,MitoTracker Green FM, Me0H, MitoTracker Orange, MitoTracker Orange,Me0H, MitoTracker Red, MitoTracker Red, Me0H, mOrange, mPlum, mRFP,mStrawberry, mTangerine, NBD-X, NBD-X, Me0H, NeuroTrace 500/525, greenfluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile Red-lipid,Nissl, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0,Oregon Green 514, Oregon Green 514 antibody conjugate pH 8.0, PacificBlue, Pacific Blue antibody conjugate pH 8.0, Phycoerythrin, PO-PRO-1,PO-PRO-1¬DNA, PO-PRO-3, PO-PRO-3-DNA, POPO-1, POPO-1-DNA, POPO-3,Propidium Iodide, Propidium Iodide-DNA, R-Phycoerythrin pH 7.5, ReAsH,Resorufin, Resorufin pH 9.0, Rhod-2, Rhod-2 Ca2+, Rhodamine, Rhodamine110, Rhodamine 110 pH 7.0, Rhodamine 123, Me0H, Rhodamine Green,Rhodamine phalloidin pH 7.0, Rhodamine Red-X antibody conjugate pH 8.0,Rhodaminen Green pH 7.0, Rhodol Green antibody conjugate pH 8.0,Sapphire, SBFI-Na+, Sodium Green Na+, Sulforhodamine 101, SYBR Green I,SYPRO Ruby, SYTO 13-DNA, SYTO 45-DNA, SYTOX Blue-DNA,Tetramethylrhodamine antibody conjugate pH 8.0, Tetramethylrhodaminedextran pH 7.0, Texas Red-X antibody conjugate pH 7.2, TO-PRO-1¬DNA,TO-PRO-3-DNA, TOTO-1-DNA, TOTO-3-DNA, TRITC, X-Rhod-1 Ca2+,YO-PRO-1¬DNA, YO-PRO-3-DNA, YOY0-1-DNA, and YOYO-3-DNA.

Other labels besides fluorescent molecules can be used, such aschemiluminescent molecules, which will give a detectable signal or achange in detectable signal upon hybridization, and radioactivemolecules. In addition, affinity labels including but not limited tobiotin, dual biotin and digoxigenin may be used.

Methods

The disclosure provides methods for using the composition comprising anucleic acid polymerase and an attenuator molecule. In one embodiment, amethod of extending a substrate polynucleotide is provided comprisingincubating the substrate polynucleotide with a composition as describedherein under conditions sufficient to allow addition of a tail sequenceto the 3′ end of the substrate polynucleotide, wherein the addition ofthe tail sequence allows association between the tail sequence and theattenuator molecule to form a complex. In some aspects, the methodfurther comprises degrading the attenuator molecule following extensionof the substrate polynucleotide. In another aspect, practice of themethods of the disclosure further comprises isolating the extendedsubstrate polynucleotide. In some aspects, the methods described hereinfurther comprise mixing a composition as described herein with thesubstrate polynucleotide and a nucleotide that is complementary to thehomopolymeric portion of the attenuator molecule. Various aspects of thedisclosure contemplate a substrate polynucleotide and/or attenuatormolecule that is partially double stranded. In addition, some aspects ofthe methods further comprise an annealing step, wherein a doublestranded polynucleotide is produced by annealing a first polynucleotideto a second polynucleotide under conditions sufficient to allow thefirst polynucleotide to associate with the second polynucleotide. Insome aspects of the disclosure the substrate polynucleotide is singlestranded RNA or DNA. In various aspects wherein the substratepolynucleotide is double stranded, each of the two free 3′ ends areextended. In other aspects, only one of the free 3′ ends of the doublestranded substrate polynucleotide is extended. In aspects wherein onlyone of the free 3′ ends of the double stranded polynucleotide isextended, it is contemplated that the other free 3′ end is preventedfrom being extended. Yet another aspect of the disclosure contemplates amethod comprising an immobilization step, wherein anattenuator/attenuator-adaptor molecule or a substrate polynucleotide orboth are immobilized to a surface. Further aspects of the disclosurecontemplate a ligating step, and still further aspects contemplate astep comprising inactivation of an enzyme. In any of the methodsdisclosed herein, it is contemplated that more than one reaction takesplace in the same reaction vessel. By way of example, the disclosurecontemplates methods wherein a tailing reaction and a ligation occurs inthe same reaction vessel.

Accordingly, the methods provided by the disclosure comprise, in variousaspects, an incubation step, a degrading step, a mixing step, anisolation step, an annealing step, an inactivating step, a ligating stepand an immobilization step. In some aspects, the method comprises anincubation step and a mixing step. In another aspect, the methodcomprises an incubation step and an isolation step. In some aspects, themethod comprises an incubation step and an inactivating step. A furtheraspect of the disclosure provides a method comprising an incubationstep, a mixing step and a ligating step. Another aspect of thedisclosure provides a method comprising an incubation step, aninactivating step and a degrading step. In a further aspect, the methodcomprises an incubation step, a mixing step, and an annealing step.Another aspect of the disclosure provides a method comprising anincubation step, a mixing step, an annealing step, a ligating step andan immobilization step. In yet another aspect, the method comprises anincubation step, a mixing step, an annealing step and an isolation step.A further aspect of the disclosure contemplates a method comprising anincubation step, a mixing step, an annealing step, a degradation stepand an isolation step. Yet another aspect of disclosure provides amethod comprising an incubation step, a mixing step, an annealing step,a degradation step, an immobilization step and an isolation step. Afurther aspect of the disclosure provides a method comprising anincubation step, a mixing step, an annealing step, an inactivating step,a degradation step, an immobilization step and an isolation step. Itwill be understood by one of skill in the art that the various steps canbe used in any combination and order, with only the mixing andincubation steps being the common feature to all methods.

Also contemplated is a method whereby NGS library preparation asdescribed herein using controlled tailing and ligation is coupled withan enrichment step for targeted NGS sequencing. In one embodiment, theinput substrate polynucleotide for controlled tailing and ligationmediated NGS library preparation is an enriched fraction of a genomeobtained by any method, including but not limited to hybridizationcapture and target-specific PCR. In another embodiment, the product of acontrolled tailing and ligation mediated NGS library as described inthis disclosure is subsequently subject to targeted enrichment by anymethod, including but not limited to hybridization capture. In analternative embodiment, the input substrate polynucleotide is firstsubject to a controlled tailing and ligation reaction to introduce afirst NGS adaptor, wherein a second step comprising a targetedenrichment by hybridization capture is performed, and in a third step, asecond NGS adaptor is introduced on the enriched DNA fraction by eithera second controlled tailing and ligation reaction or a blunt ligation ora TA ligation.

Methods of the disclosure involving controlled tailing and ligation are,in various embodiments, applied to primer-extension products oramplification products including but not limited to those amplificationproducts derived from polymerase chain reaction, isothermalamplification and RNA transcription. The methods of the disclosure canalso be applied to synthetic nucleic acids. Specifically, the methodsare applicable for sequence analysis of synthetic oligonucleotides,synthetic genes, genomic segments and genomes.

Also contemplated in this disclosure is a method that combinessimultaneous end repair with the controlled tailing and ligationreaction. End repair includes but is not limited to the followingenzymes: polynucleotide kinase, T4 DNA polymerase, uracil DNAglycosylase, APE1 endonuclease, endonuclease III (Nth), endonuclease IV,endonuclease V, endonuclease VIII, Fpg, hAAG, hOGG1, and hsMUG1. Endrepair is a separate reaction incorporated into existing NGS librarypreparation methods to repair damage induced by physical shearing of DNAas a means of DNA fragmentation within this disclosure. In this aspect,the controlled tailing and ligation reaction conditions are compatiblewith end repair reaction conditions and can be performed simultaneouslyas a single step.

Without wishing to be bound by theory, it is contemplated by thedisclosure that, in some aspects, reactions that take place in solutionare more efficient than those that involve an immobilization step. By“more efficient” is meant that the reaction in solution is completed inless time than the same reaction following an immobilization step.

Each of the method steps described above are discussed in further detailbelow.

Incubation Step

The methods of the disclosure involve incubating a substratepolynucleotide with a composition as described herein under conditionssufficient to allow addition of a tail sequence to the 3′ end of thesubstrate polynucleotide. In some aspects, an agent selected from thegroup consisting of polyethylene glycol (PEG), a polyamine, hexaminecobalt and CoC12 is used to facilitate the association of anattenuator/attenuator-adaptor molecule and a substrate polynucleotide,or to control the addition of nucleotides to the substratepolynucleotide.

Methods provided by the disclosure also include those wherein amultiplicity of nucleotides are added to the substrate polynucleotide toform the tail sequence. In some aspects, the attenuator moleculeassociates with the tail sequence over all or part of the attenuatormolecule length. In further embodiments, the attenuator moleculeassociates with the tail sequence during the process of adding the tailsequence.

In general, methods described herein also include those whereinassociation of the attenuator molecule with the tail sequence regulatesaddition of nucleotides to the polynucleotide.

With respect to the addition of nucleotides to the substratepolynucleotide, the disclosure provides methods wherein the conditionsregulate the addition of a tail sequence to the substratepolynucleotide. For example and without limitation, in one aspect theaddition of a tail sequence to the substrate polynucleotide istemperature sensitive. In one embodiment, the temperature at which thetail sequence is added to the substrate polynucleotide is at least about4° C. In further embodiments, the temperature conditions at which thetail sequence is added is at least about 4° C. to about 50° C., about 4°C. to about 40° C., about 4° C. to about 37° C., about 4° C. to about30° C., about 4° C. to about 25° C., about 4° C. to about 20° C., about10° C. to about 50° C., about 10° C. to about 40° C., about 10° C. toabout 37° C., about 10° C. to about 30° C., about 10° C. to about 25°C., about 10° C. to about 20° C., about 20° C. to about 50° C., about20° C. to about 40° C., about 20° C. to about 37° C., about 25° C. toabout 37° C., about 25° C. to about 40° C., about 30° C. to about 40°C., at least about 5° C., at least about 6° C., at least about 7° C., atleast about 8° C., at least about 9° C., at least about 10° C., at leastabout 11° C., at least about 12° C., at least about 13° C., at leastabout 14° C., at least about 15° C., at least about 16° C., at leastabout 17° C., at least about 18° C., at least about 19° C., at leastabout 20° C., at least about 21° C., at least about 22° C., at leastabout 23° C., at least about 24° C., at least about 25° C., at leastabout 26° C., at least about 27° C., at least about 28° C., at leastabout 29° C., at least about 30° C., at least about 31° C., at leastabout 32° C., at least about 33° C., at least about 34° C., at leastabout 35° C., at least about 36° C., at least about 37° C., at leastabout 38° C., at least about 39° C., at least about 40° C., at leastabout 41° C., at least about 42° C., at least about 43° C., at leastabout 44° C., at least about 45° C., at least about 46° C., at leastabout 47° C., at least about 48° C., at least about 49° C., at leastabout 50° C. or higher.

Accordingly, in certain aspects the temperature at which the incubationstep is performed is determinative of the number of nucleotides that areadded to the substrate polynucleotide. By way of example, methods areprovided wherein the length of a tail added to a substratepolynucleotide is about 10 nucleotides at 25° C., about 11 nucleotidesat 30° C., about 13 nucleotides at 37° C. and about 16 nucleotides at45° C.

In addition to the temperature at which the incubation step isperformed, another condition that regulates the addition of a tailsequence to the substrate polynucleotide is the length of time that theincubation step is allowed to progress. In general, the length of timethat the incubation step is allowed to progress is about 0.5 minutes toabout 120 minutes. In some aspects, the length of time that theincubation step is allowed to progress is at least about 0.5 minutes andup to about 1, 2 or 3 minutes; or at least about 1 minute and up toabout 2, 5 or 10 minutes; or at least about 2 minutes and up to about 5,8 or 10 minutes; or at least about 5 minutes and up to about 10, 15 or20 minutes; or at least about 10 minutes and up to about 15, 20 or 30minutes; or at least about 20 minutes and up to about 30, 40 or 60minutes; or at least about 30 minutes and up to about 40, 60 or 80minutes; or at least about 60 minutes and up to about 80, 90 or 100minutes; or at least about 90 minutes and up to about 100, 110 or 120minutes. In various embodiments, the length of time that the incubationstep is allowed to progress is about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28, about 29, about 30, about 31, about 32, about 33,about 34, about 35, about 36, about 37, about 38, about 39, about 40,about 41, about 42, about 43, about 44, about 45, about 46, about 47,about 48, about 49, about 50, about 51, about 52, about 53, about 54,about 55, about 56, about 57, about 58, about 59, about 60, about 61,about 62, about 63, about 64, about 65, about 66, about 67, about 68,about 69, about 70, about 71, about 72, about 73, about 74, about 75,about 76, about 77, about 78, about 79, about 80, about 81, about 82,about 83, about 84, about 85, about 86, about 87, about 88, about 89,about 90, about 91, about 92, about 93, about 94, about 95, about 96,about 97, about 98, about 99, about 100, about 101, about 102, about103, about 104, about 105, about 106, about 107, about 108, about 109,about 110, about 111, about 112, about 113, about 114, about 115, about116, about 117, about 118, about 119, about 120 minutes or more.

The pH at which the incubation is performed is from about 5.0 to about9.0. In one aspect, the pH is about 7.9. In some aspects, the pH atwhich the incubation is performed is at least about pH 5.0 and up toabout pH 5.1, 5.5 or 5.8; or at least about pH 5.5 and up to about pH5.8, 6.0 or 6.2; or at least about pH 6.0 and up to about pH 6.2, 6.5 or6.8; or at least about pH 6.5 and up to about pH 7.0, 7.2 or 7.5; or atleast about pH 7.5 and up to about pH 7.8, 8.0 or 8.2; or at least aboutpH 8.0 and up to about pH 8.2, 8.5 or 9.0. In various aspects, the pH atwhich the incubation is performed is about pH 5.1, about pH 5.2, aboutpH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH about 5.7,about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2,about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7,about pH 6.8, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2,about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7,about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.1, about pH 8.2,about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7,about pH 8.8, about pH 8.9, pH 9.0 or higher.

Degrading Step

In aspects of the method wherein an attenuator molecule is degradable, adegrading step optionally follows the incubation step. In one aspect, anamount of an enzyme that possesses a nucleolytic activity is added tothe reaction vessel and the mixture is incubated for an additionalperiod of time at the optimal temperature of the enzyme. In variousaspects wherein the attenuator molecule is a polynucleotide, the enzymepossessing a nucleolytic activity is selected from the group consistingof a DNA glycosylase, an apurinic/apyrimidinic endonuclease and aribonuclease. In further aspects, the ribonuclease is selected from thegroup consisting of RNase H, RNase HII, RNase A, and RNase Tl.Accordingly, and by way of example, the attenuator molecule that isdegradable comprises, in various aspects, a uracil nucleotide anddegradation occurs as a result of the activity of uracil DNAglycosylase. In another aspect, the attenuator molecule that isdegradable comprises ribonucleotides and degradation occurs as a resultof the activity of a ribonuclease. It will be understood that thenucleolytic enzyme is chosen such that the substrate polynucleotide isnot degraded with the attenuator molecule. Thus, in one aspect, anattenuator molecule that comprises ribonucleotides will be used in amethod wherein the substrate molecule comprises deoxyribonucleotides,and the nucleolytic enzyme that is used is a ribonuclease that will notdegrade the substrate polynucleotide.

The additional period of time that a reaction vessel is incubated at adesired temperature to degrade an attenuator molecule is at least about5 minutes, but is contemplated to be from about 0.5 minutes to about 60minutes or more.

Mixing Step

Methods provided herein generally comprise mixing a nucleic acidpolymerase, a substrate polynucleotide and an attenuator molecule in asuitable reaction vessel. Additional components of the mixture comprisea suitable buffer in which the nucleic acid polymerase is optimallyactive, nucleotides for addition to the substrate polynucleotide and aligase enzyme. Optionally, and according to various methods describedbelow, further components comprise potassium, CoCb, sodium, lithium,calcium manganese, tris and its derivatives, and magnesium.

Suitable reaction vessels are known to those of skill in the art andinclude, without limitation, a microcentrifuge tube or a microtiterplate.

In some aspects, more than one type of substrate polynucleotide is addedto a single reaction vessel. Accordingly, in various aspects, more thanone type of attenuator molecule, capable of associating with the morethan one type of substrate polynucleotide, may be added to a singlereaction vessel. In further aspects, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10or more types of substrate polynucleotides and attenuator moleculescapable of associating with the more than one type of substratepolynucleotide are added to a single reaction vessel. It is furthercontemplated that a reaction comprises more than one attenuatorpolynucleotide and/or more than one attenuator-adaptor molecule and/ormixtures of an attenuator polynucleotide and an attenuator-adaptormolecule. The use of more than one attenuator polynucleotide and/or morethan one attenuator-adaptor molecule enables multiplexing as well ascontrolled DNA and RNA tailing-ligation reactions by templateindependent polymerases such as deoxynucleotidyl transferase (TdT),poly(A) polymerase or poly(U) polymerase.

For nucleic acid polymerases, the amount to be added is about 1 unit(“U”) to about 1000 U per reaction. In some aspects, the amount ofnucleic acid polymerase to be added is at least about 1 U and up toabout 2, 3 or 4 U; or at least about 2 U and up to about 3, 4 or 5 U; orat least about 5 U and up to about 20, 50 or 100 U; or at least about 5U and up to about 6, 7 or 8 U; or at least about 6 U and up to about 7,8 or 9 U; or at least about 7 U and up to about 8, 9 or 10 U; or atleast about 10 U and up to about 50, 100 or 500 U; or at least about 10U and up to about 12, 15 or 18 U; or at least about 15 U and up to about18, 20 or 25 U; or at least about 20 U and up to about 50, 100 or 1000U; or at least about 20 U and up to about 25, 30 or 35 U; or at leastabout 30 U and up to about 35, 40 or 50 U; or at least about 40 U and upto about 50, 60 or 70 U; or at least about 50 U and up to about 100, 500or 1000 U; or at least about 60 U and up to about 80, 90 or 100 U; or atleast about 100 U and up to about 120, 150 or 200 U; or at least about200 U and up to about 250, 275 or 300 U; or at least about 300 U and upto about 325, 350 or 400 U; or at least about 400 U and up to about 450,500 or 550 U; or at least about 600 U and up to about 700, 800 or 900 U;or at least about 700 U and up to about 800, 900 or 1000 U. In variousaspects, the amount of nucleic acid polymerase to be added is about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 21, about 22, about 23, about 24,about 25, about 26, about 27, about 28, about 29, about 30, about 40,about 50, about 60, about 70, about 80, about 90, about 100, about 110,about 120, about 130, about 140, about 150, about 160, about 170, about180, about 190, about 200, about 210, about 220, about 230, about 240,about 250, about 260, about 270, about 280, about 290, about 300, about310, about 320, about 330, about 340, about 350, about 360, about 370,about 380, about 390, about 400, about 410, about 420, about 430, about440, about 450, about 460, about 470, about 480, about 490, about 500,about 510, about 520, about 530, about 540, about 550, about 560, about570, about 580, about 590, about 600, about 610, about 620, about 630,about 640, about 650, about 660, about 670, about 680, about 690, about700, about 710, about 720, about 730, about 740, about 750, about 760,about 770, about 780, about 790, about 800, about 810, about 820, about830, about 840, about 850, about 860, about 870, about 880, about 890,about 900, about 910, about 920, about 930, about 940, about 950, about960, about 970, about 980, about 990 or about 1000 units or more perreaction.

The nucleotides that are added to the reaction vessel will depend on thegiven application. By way of example, if the attenuator molecule is ahomopolymeric polynucleotide, then the nucleotides that are added to thereaction vessel are the nucleotides which are complementary to thenucleotide making up the homopolymeric portion of the attenuatormolecule. It is also contemplated that in various embodiments, mixturesof nucleotides are added to the reaction vessel. Thus, in someembodiments, a mixture of deoxyribonucleotides and ribonucleotides areadded to the reaction vessel (e.g., dA/rA, dT/dU/rU, dC/rC or dG/rG). Insome embodiments, the inclusion of ribonucleotides in the reactionvessel reduces additional tailing in the presence of a polymerase suchas TdT, while the inclusion of deoxynucleotides in the reaction vesselreduces additional tailing in the presence of a polymerase such aspoly(A) polymerase or poly(U) polymerase. The nucleotide concentrationwithin the reaction vessel is generally about 0.1 mM, but in variousaspects is between about 0.01 to about 5 mM.

As described above, some embodiments of the methods include a ligaseenzyme. For the ligase enzyme, the amount to be added is between about0.1 to about 1000 U per reaction.

For magnesium, it is contemplated that the amount to be added is fromabout 1 mM to about 100 mM per reaction. In various aspects, the amountof potassium to be added is about 1 mM to about 10 mM, or about 2 mM toabout 20 mM, or about 10 mM to about 100 mM.

Isolating Step

In some embodiments, the substrate polynucleotide is isolated. Isolationof the substrate polynucleotide is performed by any method known andunderstood by one of skill in the art. In one aspect, isolation of thesubstrate polynucleotide is performed by immobilization of the substratepolynucleotide as described herein. In another aspect, ligand-coupledbeads or microspheres are used to specifically associate with thesubstrate polynucleotide and facilitate its isolation. By way ofexample, a substrate polynucleotide that was tailed with a homopolymericadenine sequence can be isolated using a poly-dT-coupled bead. In otheraspects, isolation of the substrate polynucleotide is performed byprecipitation, gel filtration or spin-column microcentrifugation of thesubstrate polynucleotide.

Immobilization Step

In some aspects, the attenuator molecule and/or the substratepolynucleotide is covalently or non-covalently coupled to a support.Coupling chemistries and selection of support materials well known inthe art are contemplated. For example, supports include those made allor in part of glass, silica, metal, plastic, fiber, resin, and polymers.Exemplary polymers include for example and without limitation cellulose,nitrocellulose, polyacetate, polycarbonate, polystyrene, polyester,polyvinyldifluorobenzene, nylon, carbon fiber or any other suitablepolymer material. In certain related embodiments one or a plurality ofthe attenuator molecules and/or substrate polynucleotides describedherein may be provided as an array immobilized on a solid support, whichincludes any of a number of well known configurations for spatiallyarranging such molecules in an identifiable (for example and withoutlimitation, addressable) fashion. Immobilization, in various aspects,involves biotinylated attenuator-adaptor molecules and streptavidin(avidin) coated surfaces (for example and without limitation, tubes,beads or magnetic beads). The skilled artisan will be familiar withvarious compositions and methods for making and using arrays of suchsolid-phase immobilized attenuator molecule and/or substratepolynucleotide arrays.

Inactivating Step

In some aspects, following incubation of the reaction vessel comprisingthe components of the reaction, the reaction vessel is further incubatedat a higher temperature to inactivate the nucleic acid polymerase. Insome aspects, the further incubation is performed for at least about 1minute and up to about 2, 5 or 10 minutes; or at least about 5 and up toabout 10, 20 or 30 minutes; or at least about 10 and up to about 15, 20or 30 minutes; or at least about 15 and up to about 20, 25 or 30minutes. In some embodiments, the further incubation is performed forabout 1 minute to about 30 minutes. In various aspects, the furtherincubation is performed for about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about28, about 29, about 30 minutes or more.

The higher temperature at which the further incubation is performed isfrom about 60 C to about 100° C. In some aspects, the temperature atwhich the further incubation is performed is at least about 60° C. andup to about 62° C., 65° C. or 68° C.; or at least about 60° C. and up toabout 65° C., 70° C. or 75° C.; or at least about 60° C. and up to about70° C., 75° C. or 80° C.; or at least about 70° C. and up to about 75°C., 80° C. or 85° C.; or at least about 70° C. and up to 80° C., 90° C.or 100° C. In various aspects, the temperature at which the furtherincubation is performed is about 61° C., about 62° C., about 63° C.,about 64° C., about 65° C., about 66° C., about 67° C., about 68° C.,about 69° C., about 70° C., about 71° C., about 72° C., about 73° C.,about 74° C., about 75° C., about 76° C., about 77° C., about 78° C.,about 79° C., about 80° C., about 81° C., about 82° C., about 83° C.,about 84° C., about 85° C., about 86° C., about 87° C., about 88° C.,about 89° C., about 90° C., about 91° C., about 92° C., about 93° C.,about 94° C., about 95° C., about 96° C., about 97° C., about 98° C.,about 99° C., about 100° C. or higher.

Ligating Step

In some aspects of the methods that are provided, the reaction is suchthat tailing of the substrate polynucleotide occurs simultaneously withsubstrate polynucleotide ligation to the adaptor molecule. In theseaspects, a mixture comprising the nucleic acid polymerase, substratepolynucleotide, attenuator-adaptor molecule, buffer, and ligase enzymeare all present in a single reaction vessel (see, for example andwithout limitation, Example 9). Incubation of the mixture to producetailing of the substrate polynucleotide and its ligation to theattenuator-adaptor molecule is identical to the methods described abovefor tailing alone. In various aspects, the ligase enzyme is a DNA ligaseor a RNA ligase. Attenuator molecules that are immobilized have beendescribed herein. In some aspects of the methods, the immobilizedattenuator molecule is ligated by a DNA or RNA ligase to apolynucleotide during addition of a tail sequence to the polynucleotidemolecule.

For a ligase enzyme, the amount to be added is about 0.1 unit (“U”) toabout 1000 U per reaction. In some aspects, the amount of ligase enzymeto be added is at least about 0.1 U and up to about 0.5, 1, 2, 3 or 4 U;or at least about 1 U and up to about 3, 4 or 5 U; or at least about 5 Uand up to about 20, 50 or 100 U; or at least about 5 U and up to about6, 7 or 8 U; or at least about 6 U and up to about 7, 8 or 9 U; or atleast about 7 U and up to about 8, 9 or 10 U; or at least about 10 U andup to about 50, 100 or 500 U; or at least about 10 U and up to about 12,15 or 18 U; or at least about 15 U and up to about 18, 20 or 25 U; or atleast about 20 U and up to about 50, 100 or 1000 U; or at least about 20U and up to about 25, 30 or 35 U; or at least about 30 U and up to about35, 40 or 50 U; or at least about 40 U and up to about 50, 60 or 70 U;or at least about 50 U and up to about 100, 500 or 1000 U; or at leastabout 60 U and up to about 80, 90 or 100 U; or at least about 100 U andup to about 120, 150 or 200 U; or at least about 200 U and up to about250, 275 or 300 U; or at least about 300 U and up to about 325, 350 or400 U; or at least about 400 U and up to about 450, 500 or 550 U; or atleast about 600 U and up to about 700, 800 or 900 U; or at least about700 U and up to about 800, 900 or 1000 U. In various aspects, the amountof ligase enzyme to be added is about 0.2, about 0.3, about 0.4, about0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 40, about50, about 60, about 70, about 80, about 90, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 260, about 270, about 280, about 290, about 300, about 310,about 320, about 330, about 340, about 350, about 360, about 370, about380, about 390, about 400, about 410, about 420, about 430, about 440,about 450, about 460, about 470, about 480, about 490, about 500, about510, about 520, about 530, about 540, about 550, about 560, about 570,about 580, about 590, about 600, about 610, about 620, about 630, about640, about 650, about 660, about 670, about 680, about 690, about 700,about 710, about 720, about 730, about 740, about 750, about 760, about770, about 780, about 790, about 800, about 810, about 820, about 830,about 840, about 850, about 860, about 870, about 880, about 890, about900, about 910, about 920, about 930, about 940, about 950, about 960,about 970, about 980, about 990 or about 1000 units or more perreaction.

Therapeutic Applications

In addition, the disclosure also contemplates therapeutic applicationsof the attenuated substrate polynucleotide for the control of cellularand viral proliferation. Therapeutic application include but are notlimited to antisense regulation of gene expression. Poly(A) polymerasesin eukaryotes are responsible for the addition of poly(A) tails duringmessenger RNA processing. The poly(A) tails of the resulting mRNAs servemultiple functions. They are required for the transport from the nucleusto the cytoplasm, they stimulate the efficiency of protein synthesis andthey stabilize mRNA. Polyadenylation of RNA in bacteria plays asignificant role in RNA decay. Addition of poly(U) tails in eukaryotesis less understood but may control the degradation of certain RNAs.Synthetic attenuator molecules can potentially be used as antisensemolecules to inhibit or limit poly(A) and poly(U) tailing within thecell and thus establish control of cellular and viral proliferation.

Kits

The disclosure provides kits for controlled and limited nucleic acidtailing by a nucleic acid polymerase.

A kit provided by the disclosure comprises an attenuator/optionalattenuator-adaptor molecule as described herein (including optionalsolid phase immobilized attenuator-adaptor molecules), a nucleic acidpolymerase, optionally a ligase, a glycosylase and ancillary reagentssuch as appropriate buffers, wash solutions, indicators and detectionmedia, depending on the particular assay configuration to be practiced.In some aspects, attenuator molecules are premixed with the nucleic acidpolymerase or provided in a separate tube.

Examples of such kits include but are not limited to the following.

TdT-Mediated DNA Tailing

This kit comprises the following components. For a poly (dA) tailingkit: TdT enzyme supplemented with 3′-blocked linear or circular poly(dT), poly(dU) or poly(U) attenuator molecule. For a poly (dT) tailingkit: TdT enzyme supplemented with 3′-blocked linear or circular poly(dA) or poly(A) attenuator molecule. For a poly (dG) tailing kit: TdTenzyme supplemented with 3′-blocked linear or circular poly (dC) orpoly(C) attenuator molecule. For a poly (dC) tailing kit: TdT enzymesupplemented with 3′-linear linear or circular poly (dG) or poly(G)attenuator molecule.

Poly(A) and Poly(U)-Polymerase-Mediated RNA Tailing

A poly(A) tailing kit comprises: Poly(A) polymerase supplemented withpoly (dT), poly(dU) or poly (U) attenuator molecule. A Poly (U) tailingkit comprises: Poly(U) polymerase supplemented with poly (dA), or poly(A) attenuator molecule.

Additional kits comprise reagents for single-reaction tailing andadaptor ligation both for DNA and RNA substrates, and reagents forsingle reaction tailing-ligation-immobilization both for DNA and RNAsubstrates. Other kits can introduce barcodes to DNA and RNA molecules.Still other kits can convert DNA and RNA substrates into libraries fornext generation sequencing. In one embodiment, a NGS library preparationkit is provided comprising materials for performing (i) a controlledtailing reaction; (ii) end repair; (iii) primer extension; and (iv)blunt end or TA ligation or a second controlled tailing and ligation.

Provided below in the Examples section are specific applications usingthe compositions and methods described by the disclosure. It will beunderstood that these applications are provided by way of example only,and are not limiting in any way.

EXAMPLES Example 1 Attenuated, TdT-Mediated Poly(dA) DNA Tailing in thePresence of Long (>20b) Complementary Poly(dT) Polynucleotide

Phase 1: Non-attenuated and fast TdT-mediated poly(dA) tailing of a DNAprimer occurs at 37° C. until the size of the tail reaches a criticalsize that is capable of forming a stable complex with the complementaryattenuator molecule containing the long (dT)30 sequence (SEQ ID NO: 62).Tailing of the attenuator molecule is prevented by placing a blockinggroup at the 3′ end of the attenuator molecule (for example and withoutlimitation phosphate, dideoxynucleotide, amino group, inverted dT) orseveral ribonucleotides, or by using circular attenuator molecules.

Phase 2: Formation of a complex between the attenuator polynucleotide(dT)30 (SEQ ID NO: 62) and the poly(dA) tail results in a significantreduction of the poly(dA) synthesis. Each subsequent dA base added bythe TdT enzyme increases the length and stability of the duplex, thusleading to almost complete inhibition of the poly(dA) synthesis by theTdT enzyme (FIG. 1).

Kinetics of Poly(dA) Tailing in the Presence of Long Poly(dT) AttenuatorMolecule

In some embodiments of the methods, TdT-mediated dA tailing at 37° C. inthe presence of a long attenuator molecule produces relatively shorttails (−13 b) with very narrow size distribution as depicted in FIG. 38.

Attenuated TdT-Mediated Poly(dA) Tailing with Short (12-14b)Complementary Poly (dT) Polynucleotides

In further embodiments, a method is provided wherein non-attenuated andfast TdT¬mediated poly(dA) tailing of a substrate DNA primer occurs inphase 1 (FIG. 2). Formation of a complex between the attenuatorpolynucleotide and poly(A) tail and reduction of the poly(dA) synthesisrate in phase 2a. At this phase every additionally added dA-basestabilizes the complex more until the tail length reaches the fulllength of the attenuator molecule and forms a blunt duplex end. Thereaction never goes into Phase 2b as in alternative embodiments of themethods (see above) because of a limited size of the attenuatormolecule.

In phase 3 (FIG. 3), slow poly(dA) tailing of a blunt-ended substratethen occurs until creation of a 3′ single-stranded poly(dA) overhangcontaining 3-4 dA bases. Phases 1 through 3 are then repeated.

Kinetics of Poly(dA) Tailing in the Presence of Short Poly(dT)Attenuator Molecule

In some aspects, TdT-mediated DNA dA tailing in the presence of a shortattenuator molecule results in the synthesis of DNA molecules where thepoly(dA) tails have a discrete, ladder-like size distribution with thelength of tail having, for example and without limitation, multiples of13 bases (13, 26, 39, etc.) (FIG. 3). In another embodiment, a method iscontemplated wherein attenuated TdT-mediated poly(dA) tailing isperformed with a degradable attenuator polynucleotide containing dUbases (FIG. 4).

Methods provided by the disclosure also include the attenuatedTdT-mediated poly(dT), poly(dG) and poly(dC) tailing with a long (about20-30 bases) complementary attenuator polynucleotide (see FIG. 5). Alsoprovided is a method of attenuated TdT-mediated poly(dA), poly(dT),poly(dG) and poly(dC) tailing with degradable attenuatorribo¬polynucleotides (FIG. 6).

Controlled RNA Tailing by Poly(A) and Poly(U) Polymerases

The methods described herein relating to the attenuation and the controlof TdT-mediated homopolymeric DNA tailing are also contemplated to beapplied to an enzymatic reaction catalyzed by poly (A) or poly (U)polymerase that add poly(A) and poly(U) sequences to RNA templates.Similar to methods relating to DNA, 3′-blocked linear poly(U) andpoly(A), non-blocked poly (dU) and poly(dA) molecules, and poly(U),poly(A), poly (dU), poly(dA) poly (dU) and poly(dA) molecules, andpoly(U), poly(A), poly (dU), poly(dA) circles can be used as attenuatorsof poly(A) and poly(U) polymerases.

Controlled tailing of RNA by poly(A) and poly(U) polymerases with C andG ribonucleotides, in various aspects, requires a corresponding DNA orRNA attenuator molecule.

FIG. 7 depicts attenuated poly (A)-polymerase-mediated poly (rA) tailingof RNA substrates using a complementary DNA poly (dT)30 polynucleotide(SEQ ID NO: 62). In phase 1 (top portion of FIG. 7), non-attenuated andfast poly(A)-polymerase-mediated poly (A) tailing of an RNA primer isshown. In phase 2 (following addition of a poly (rA) tail), formation ofa stable complex between the attenuator polynucleotide poly(dT)30 (SEQID NO: 62) and the poly (A) tail results in a significant reduction oreven complete inhibition of the poly (A) synthesis.

Another aspect of the methods provides attenuated poly(U)-polymerase-mediated poly (rU) tailing of RNA substrates usingcomplementary DNA poly (dA)30 (SEQ ID NO: 68) polynucleotide (FIG. 8).The top portion of FIG. 8 depicts phase 1 of such methods, whereinnon-attenuated poly(U)-polymerase-mediated poly (U) tailing of an RNAprimer takes place. In phase 2 (following addition of a poly (rU) tail),formation of a stable complex occurs between the attenuatorpolynucleotide poly(dA)30 (SEQ ID NO: 68) and the poly (U) tail resultsin a significant reduction or even complete inhibition of the poly (U)synthesis.

Use of Controlled, Size-Limited Tailing for Adaptor (Barcode) Attachmentto One End of DNA or RNA Fragments and Immobilization to a Solid Support

Previously described attenuator molecules are degradable ornon-degradable homopolymeric molecules complementary to tails producedby, for example and without limitation, TdT, poly(A) or poly(U)polymerases. Below is introduced a class of attenuator molecules that inaddition to their homopolymeric 3′-domain have a single-stranded ordouble-stranded domain at the 5′ portion. These domains are used tointroduce an adaptor sequence downstream of the tail region bypolymerization or ligation reaction. An advantage of the ligationreaction is that it is coupled with the non-template homopolymerictailing reaction in a single-tube, single-step reaction. Use of suchtailing-ligation reactions provides a simple and efficient way forcreation of DNA, RNA or cDNA libraries with one or two adaptors withapplication for sample preparation from genomic DNA and RNA nextgeneration sequencing (NGS) applications. A schematic of 3′-end adaptorattachment to single-stranded DNA or RNA molecules using a limitedtailing reaction is provided in FIG. 9. Part A of FIG. 9 depicts alimited tailing-ligation reaction. In such a reaction, single strand(ss) DNA or RNA is incubated with a template-independent polymerase andligase in the presence of an attenuator-adaptor molecule that ispartially double stranded, where the 3′-blocked single-stranded poly(T)or poly (dT) portion of the attenuator-adaptor serves as an attenuatorand the 5′-phosphorylated, double-stranded portion of theattenuator-adaptor serves as an adaptor. A limited poly(dA) or poly(A)stretch is then added to the polynucleotide via a template-independentpolymerase, and forms a duplex with the single-stranded portion of theattenuator-adaptor. The adaptor portion of the attenuator-adaptor isthen ligated to the attenuated polynucleotide via the 5′ phosphatepresent on the adaptor molecule. The controlled tailing and ligationreactions occur in a closed-tube format. The adaptor molecule optionallyfurther comprises a tag (for example and without limitation, biotin).The ligated molecule is then optionally immobilized tostreptavidin-coated magnetic beads to facilitate isolation.

Part B of FIG. 9 depicts a limited tailing-polymerase-extensionreaction. In such a reaction, DNA or RNA is incubated with atemplate-independent polymerase in the presence of an attenuator-adaptormolecule that is single stranded. A poly(dA) or poly(A) stretch is thenadded to the polynucleotide via a template-independent polymerase, andthe presence of a DNA polymerase will allow for extension across the DNAor RNA molecule, thereby creating a double stranded product. Thecontrolled tailing and extension reactions can be done in a closed-tubeformat. In this case, the deoxynucleotide triphosphate (dNTP) mix mustinclude heat¬activatable dTTP, dCTP and dGTP (CleanAmp nucleotides,TriLink Bio Technologies, San Diego) and standard dATP, and the 3′ endof a single stranded attenuator-adaptor must also contain aheat-activated base. As a result, controlled attenuated tailing wouldoccur at 37° C. when only dATP is available and the other nucleotidesand 3′ end of the attenuator-adaptor remains blocked. After heating themixture at 95° C., the remaining nucleotides become activated and the 3′end of the attenuator-adaptor becomes extendable. The adaptor moleculeoptionally further comprises a tag (for example and without limitation,biotin). The product molecule is then optionally immobilized tostreptavidin-coated magnetic beads to facilitate isolation.

In a further aspect of the disclosure is provided a method for covalentimmobilization of single-stranded DNA and RNA to a solid support using alimited tailing reaction (FIG. 10). Part A of FIG. 10 depictsimmobilization by 3′-end using a limited tailing-ligation reaction. Insuch a reaction, a DNA or RNA molecule is incubated with atemplate-independent polymerase and a ligase in the presence of a 3′-endcovalently immobilized attenuator-adaptor molecule that is partiallydouble stranded, where the 3′ end blocked single-stranded poly(T) orpoly(dT) portion serves as an attenuator and the 5′ phosphorylateddouble stranded portion serves as an adaptor. A limited poly(dA) orpoly(A) stretch is then added to the substrate by a template-independentpolymerase and the substrate forms a duplex with the single strandedportion of the attenuator-adaptor. The adaptor portion of theimmobilized attenuator-adaptor is then ligated to the attenuatedsubstrate polynucleotide via the 5′ phosphate present on the adaptormolecule resulting in an immobilized DNA or RNA molecule. The controlledtailing, ligation and immobilization reactions occur in a closed-tubeformat.

Part B of FIG. 10 depicts immobilization by 5′-end using limitedtailing-polymerase-extension reaction, which is a further aspect of thedisclosure. In such a reaction, a DNA or RNA molecule is incubated witha template-independent polymerase in the presence of a 5′-end covalentlyimmobilized attenuator-adaptor molecule. A poly(dA) or poly(A) stretchis then added by a template-independent polymerase, and the presence ofa DNA polymerase allows for extension across the DNA or RNA molecule,thereby creating an immobilized double stranded product. The controlledtailing, extension and immobilization reactions can be done in aclosed-tube format. In this case the dNTP mix must includeheat-activatable dTTP, dCTP and dGTP (CleanAmp nucleotides, TriLink BioTechnologies, San Diego) and standard dATP, and the 3′ end of animmobilized single stranded attenuator-adaptor must also contain aheat-activated base. As a result, controlled attenuated tailing wouldoccur at 37° C. when only dATP is available and the other nucleotidesand 3′ end of the immobilized attenuator-adaptor remain blocked. Afterheating the mixture at 95° C., the remaining nucleotides becomeactivated and the 3′ end of the immobilized attenuator-adaptor becomesextendable.

In some aspects, in the case of DNA substrates, simultaneoustailing-ligation reactions (FIG. 9, part A, and FIG. 10, part A) willinvolve a TdT enzyme and E. coli DNA ligase (without being bound bytheory, it is contemplated that in some embodiments, the ATP requiredfor T4 DNA ligase would block a TdT-tailing process). In the case of RNAsubstrates, simultaneous tailing-ligation reactions (FIG. 9, part A, andFIG. 10, part A) involves poly(A) and T4 DNA ligase or poly (U)polymerase. In some aspects, however, the presence of ATP results inmixed poly(U/A) tailing.

In the case of DNA substrates, tailing-extension reactions (FIG. 9, partB and FIG. 10, part B) are executed by a TdT enzyme and a mesophilic orthermophilic DNA polymerase. In the case of RNA substrates,tailing-extension reactions (FIG. 9, part B and FIG. 10, part B) areexecuted by poly(A) or poly(U) polymerase and a DNA polymerase withreverse transcriptase activity. Polymerases contemplated for use in themethods and compositions of the disclosure have been described hereinabove.

Use of Controlled, Size-Limited Tailing for Adaptor Attachment to BothEnds of a DNA Library with and without Immobilization to a Solid Support

In the case of adaptor ligation to double-stranded substrates (FIG. 11),the method involves a high concentration of a DNA ligase (for exampleand without limitation, T4 DNA ligase) and a blunt-end adaptor.Alternatively, the adaptor has a single dT-base 3′-overhang if thesecond DNA strand of the substrate nucleic acid was synthesized by apolymerase without 3′ proofreading activity. In some aspects, suchpolymerases add an additional dA base to the 3′ end of DNA.

In the case of a single-stranded DNA substrate (such substrates can becovalently immobilized by their 5′ end or through a biotin-streptavidininteraction as shown FIGS. 12 and 13, or may be free in solution),attachment of the second adaptor can involve processes shown in FIG. 9that utilize limited tailing coupled either with the ligation process(FIG. 12) or with a polymerization process (FIG. 13).

Example 2 Length of Poly(dA) Sequences Introduced by TdT Enzyme in thePresence of Long Poly(dT) Attenuator Molecule

FIG. 14 shows the calculated dependence (open circles) between thelength of the poly (dA)/poly (dT) duplex and its melting temperature,and it was concluded that the expected size of the attenuated poly(dA)tail varies as a function of the reaction temperature but is limited bya range of 10-16 bases.

Controlled Poly(dA) Tailing of Single-Stranded DNA PolynucleotideTemplate by TdT Enzyme in the Presence of a Long (Degradable) AttenuatorMolecule

Materials:

Substrate polynucleotide 10-001 (Table 1) Long degradable attenuatorpolynucleotide 10-003 (Table 2) DNA polynucleotide size marker:equimolar mix of 5 polynucleotides 10-001, 10-099, 10-100, 10-101 and10-102 (Table 4) TdT enzyme (New England BioLabs, Cat # M0315S, 20 U/μl)1 × TdT buffer: 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Mg ·acetate, 0.25 mM CoCl₂, pH 7.9 at 25° C. USER enzyme (New EnglandBioLabs, Cat # M5505S, 1 U/μl)

Method:

Poly(dA) tailing reactions were performed in 5 pi reaction volumes,containing 1×TdT buffer, 0.1 mM dATP, 4 pmol of the substratepolynucleotide 10-001, 10 U TdT enzyme and 0 or 20 pmol of theattenuator polynucleotide 10-103 at 37° C. for 1, 5, 15, 30, and 60minutes, followed by 10 min incubation at 70° C. to inactivate the TdTenzyme. 0.25 U of the USER enzyme were added and incubated 5 minutes at37° C. Samples were boiled in formamide loading buffer and run on apre-casted 15% TBE-Urea polyacrylamide gel (Invitrogen, Cat#EC68852Box), stained with SYBR Gold stain (Invitrogen, Cat #S11494),visualized on a Dark Reader light box (Clare Chemical Research), andphotographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuated poly(dA)tailing reactions by the TdT enzyme are shown on FIG. 15. Lanes 1, 2, 3,4 and 5 show the products of tailing reaction after 1, 5, 10, 15 and 30minutes of incubation with TdT enzyme in the presence of attenuator10-103; lanes 7, 8, 9, 10 and 11 show the products of tailing reactionafter 1, 5, 10, 15 and 30 minutes of incubation with TdT enzyme in theabsence of attenuator; lane 6—DNA polynucleotide size marker. In bothcases the tailing reaction was completed within 30 minutes. In theabsence of attenuator molecule the TdT enzyme added to the substratepolynucleotide very long and heterogeneous in size poly(dA) tails. Inthe presence of the attenuator molecule, the size of the added poly(dA)tails was very discrete, its distribution was very narrow, with amaximum at about 12-13 bases (long attenuator molecule degraded by USERenzyme was not visible on the gel because degradation products did notexceed 5 bases). It is interesting to note that the melting temperature(or stability) of the complex formed by the attenuator molecule 10-103and the poly(dA) tail containing 13 dA bases (SEQ ID NO: 66) was about37.6° C., that is very close to the reaction temperature 37° C. Theabsence of any products with tails exceeding 13 bases indicated that theaddition of dA bases was strongly inhibited by the attenuator moleculeabove this limit.

Conclusions:

Complete attenuation of the poly(dA) tailing was achieved using long (40b) poly (dT) molecules (SEQ ID NO: 85). The length of poly(dA) tailsadded by the TdT enzyme in the presence of long attenuator moleculesconstituted about 12-13 dA bases with extremely narrow size distributioncontrasting several hundred dA bases added in the absence of attenuatormolecules. Attenuator molecules containing dU bases were degraded aftercompletion of the tailing reaction using USER enzyme to simplifydownstream utilization of the dA-tailed DNA substrates.

Example 3 Controlled Poly(dA) Tailing of Single-Stranded DNAPolynucleotide Template by TdT Enzyme in the Presence of a ShortAttenuator Molecule

Materials:

Substrate polynucleotide 10-001 (Table 1) Short attenuatorpolynucleotides: 10-130, 10-131, 10-132, 10-133, 10-134 and 10-135(Table 2) Two ribo-U nucleotides at the ends of short attenuatormolecules were added to prevent tailing of the attenuator molecules bythe TdT enzyme Long degradable attenuator polynucleotide 10-103 (Table2) DNA polynucleotide size marker: equimolar mix of polynucleotides10-001, 10-099, 10-100, 10-101, and 10-102 (Table 4)

25 bp ladder DNA size marker (Invitrogen, Cat #10488-022) TdT enzyme(New England BioLabs, Cat # M031S, 20 U/μl) 1 × TdT buffer: 20 mMTris-acetate, 50 mM potassium acetate, 10 mM Mg · acetate, 0.25 mMCoCl₂, pH 7.9 at 25° C. USER enzyme (New England BioLabs, Cat # M55058,1 U/μl)

Method:

Poly(dA) tailing reactions were performed in 5 μl reaction volumes,containing 1×TdT buffer, 0.1 mM dATP, 4 pmol of the substratepolynucleotide 10-001, 10 U TdT enzyme, 60 pmol of the short attenuatorpolynucleotides 10-130-10-135 or 20 pmol of the long attenuatorpolynucleotide 10-103 at 30° C. for 30 minutes, followed by 10 minuteincubation at 70° C. to inactivate the TdT enzyme. Controlled reactionswith the substrate 10-001 were also conducted in the presence of theattenuator molecule 10-133 for 0, 30, 60, 90 and 120 minutes. 0.25 U ofthe USER enzyme was added to the tube containing the long attenuatormolecule 10¬103 and incubated 5 minutes at 37° C. Samples were boiled informamide loading buffer and run on a pre-casted 15% TBE-Ureapolyacrylamide gel (Invitrogen, Cat #EC68852Box), stained with SYBR Goldstain (Invitrogen, Cat #S11494), visualized on a Dark Reader light box(Clare Chemical Research), and photographed using a digital camera.

Results:

Electrophoretic analysis of products of attenuated poly(dA) tailingreactions by the TdT enzyme using attenuator molecules of differentlength and stability are shown on FIG. 16a . Lane 2 shows untailedtemplate polynucleotide-substrate 10-001, lanes3-10—polynucleotide-substrate 10-1001 after TdT tailing. Lane 3 showsuncontrolled tailing product, lane 4—controlled tailing product in thepresence of long attenuator molecule 10-103, and lanes 5, 6, 7, 8, 9,and 10—controlled tailing products on the presence of short attenuatormolecules 10-130, 10-131, 10-132, 10-133, 10-134 and 10-135,respectively. Lanes 1 and 11 show DNA polynucleotide and 25 bp laddersize markers, respectively. FIG. 16b shows the kinetics of the tailingreaction in the presence of attenuator molecule 10-133, lane 1—substrate10-001, lanes 2, 3, 4, and 5—tailing products after incubation with theTdT enzyme for 30, 60, 90, and 120 min, respectively. As in Example 2,in the presence of long attenuator molecule 10-103 the size of addedpoly(dA) tails was very discrete, its distribution was very narrow, withmean value at about 12-13 bases. The effect of short attenuatormolecules was more complex and depended on their size. Attenuatormolecules 10-130 and 10-131 with T7 rUrU and T8 rUrU stretches (SEQ IDNO: 88) (capable of forming complexes with the poly (dA) tail withmelting temperatures Tm, =14.5° C. and 10° C., respectively) onlyslightly reduced the average size of poly(dA) tails while their sizedistribution still remained very broad (FIG. 16a , lanes 5 and 6).Attenuator molecule 10-132 with a T9 rUrU stretch (SEQ ID NO: 89),capable of forming a complex with the poly (dA) tail with meltingtemperature Tm=24.7° C., produced a predicted ladder of bands with anincrement of approximately 15 bases (FIG. 16a , lane 7). The ladderbecame more prominent when incubation time was increased up to 90-120minutes as seen from FIG. 16b , lanes 4 and 5. Such kinetics ofattenuated tailing with short attenuator molecules was theoreticallypredicted and discussed herein. Attenuator molecules 10-133, 10-134 and10-135 (capable of forming complexes with the poly (dA) tail withmelting temperatures Tm=28.6° C., 32° C. and 35° C., respectively)produced a single discrete band with a size that gradually decreasedfrom 15 to 12 bases upon increase of the attenuator size (FIG. 16a ,lanes 8-10). Attenuator molecule 10-135, with a total number of 14bases, was capable of forming a complex with the poly(dA) tail withTm=35° C. and had the same effect as the long 40-base attenuatormolecule 10-103. 12-base length for poly (dA) tails (SEQ ID NO: 81)observed at reaction temperature 30° C. is one base lower than the13-base size (SEQ ID NO: 73) observed at reaction temperature 37° C., inagreement with the expected increased stability of complexes of shorterlength at the lower reaction temperature. Attenuator molecules10-130-10-135 were seen at the bottom of gel shown in FIG. 16a , lanes5-10).

Conclusions:

Complete attenuation of the poly(dA) tailing was achieved using short(12-14 base) poly (dT) molecules (SEQ ID NO: 90) blocked at the 3′ endby 1-2 ribonucleotides, a phosphate group or other modificationspreventing TdT tailing of the attenuator molecule. The length ofpoly(dA) tails added by the TdT enzyme in the presence of attenuatormolecules was controlled by the reaction temperature and the size ofattenuator molecules. Attenuated poly(dA) tails have a very narrow sizedistribution (+/−1 base) with the mean value varying from 11 to 15bases. Prolonged incubation with TdT in presence of attenuatorscontaining approximately 12 dT bases (SEQ ID NO: 91) resulted in repeatswith an increment of 12-15 dA bases (SEQ ID NO: 92).

Example 4 Both Controlled and Uncontrolled Poly(dA) Tailing ofDouble-Stranded DNA Polynucleotide Templates by TdT Enzyme isInefficient and Displays Strong Sequence Bias

Materials:

Double-stranded DNA substrate with GC-rich blunt end formed bypolynucleotides 10-105 and 10-106 (Table 1 and Table 5): Double-strandedDNA substrate with AT-rich blunt end formed by polynucleotides 10-107and 10-108 (Table 1 and Table 5): Double-stranded DNA substrate with3′-overhanging end (3 bases) formed by polynucleotides 10-105 and 10-109(Table 1 and Table 5): Double-stranded DNA substrate with 3′-recessedend (3 bases) formed by polynucleotides 10-105 and 10-110 (Table 1 andTable 5): Long degradable attenuator polynucleotide 10-103 (Table 2): 25bp ladder DNA size marker (Invitrogen, Cat #10488-022): TdT Enzyme (NewEngland Biolabs, Cat# M0315S, 20 U/μL): 1 × TDT Buffer: 20 mMTris-acetate, 50 mM potassium acetate, 10 mM Mg · acetate, 0.25 mMCoCl₂, pH 7.9

Method:

Double-stranded DNA templates were prepared by annealing thepolynucleotide pairs 10-105/10-106, 10-107/10-108, 10-105/10-109, and10-105/10-110. Specifically, after boiling, the mixed polynucleotideswere allowed to cool slowly to room temperature in 10 mM Tris-HClcontaining 0.1 mM EDTA and 50 mM NaCl. Poly(dA) tailing reactions wereperformed in a 5 uL reaction volume containing 1×TdT buffer, 0.1 mMdATP, 1 pmol of the substrate polynucleotide pair 10-105/106, 105/109,105/110, or 107/108, 10 U TdT enzyme, 0.5 t_IL of 2.5 mM CoC12, and 0 or20 pmol of the attenuator polynucleotide 10-103 at 37° C. for 60minutes, followed by 10 minutes of incubation at 70° C. to inactivateTdT enzyme. 0.25 U of the USER enzyme were added and incubated 5 minutesat 37° C. Samples were boiled in formamide loading buffer and run on aprecast 15% TBE-Urea gel (Invitrogen Cat #EC68852BOX), stained with SYBRGold (Invitrogen Cat #S11494), visualized on a Dark Reader light box(Clare Chemical Research), and photographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuated poly(dA)tailing reactions by the TDT enzyme are shown in FIG. 17. Lanes 1-4shows the tailing products in the presence of attenuator molecule, lanes6-10—in the absence of attenuator molecule, lane 5-25 base pairs laddersize marker, lane 6—uncontrolled tailing of the single-strandedpolynucleotide-substrate 10-001. All double-stranded templates exhibitedless efficient TdT tailing than single-stranded templates.Double-stranded constructs with a 3′ overhang (FIG. 17, lanes 2 and 8)were better templates for tailing reaction than double-strandedconstructs with recessed end (FIG. 17, lanes 3 and 9) or blunt end (FIG.17, lanes 1, 4, 7 and 10). The AT-rich blunt-ended construct (FIG. 17,lanes 4 and 10) was more efficiently tailed than the correspondingGC-rich construct (FIG. 17, lanes 1 and 7). Controlled tailing was morepronounced for the construct with the 3′ overhang (FIG. 17, lane 2)although it did not go to completion.

Conclusions:

Tailing of blunt-ended double stranded DNA (dsDNA) occurred much moreslowly than tailing of dsDNA with a 3′ overhang. AT rich blunt ends weretailed more efficiently than GC rich ends. Without wishing to be boundby theory, this may have been due to increased “breathing” of the 3′ endof an AT rich sequence, allowing it to behave somewhat like singlestranded DNA (ssDNA). Double stranded DNA (dsDNA) with a recessed endleads to little if any tailing. Controlled tailing can be observed andit is more efficient for double-stranded DNA molecules with the 3′single-stranded overhangs.

Example 5 Controlled Poly(dA) Tailing of Single-Stranded DNAPolynucleotide Templates by TdT Enzyme in the Presence of AttenuatorMolecules Displays No Sequence Bias

Materials:

Substrate polynucleotide 10-001 (Table 1) Substrate polynucleotide withrandom sequences 10-127, 128, 129, and 139 (Table 1) Long degradableattenuator polynucleotide 10-103 (Table 2)

TdT Ezyme (New England Biolabs, Cat# M0315S, 20 U/μL) 1 × TdT Buffer: 20mM Tris-acetate, 50 mM potassium acetate, 10 mM Mg · acetate, 0.25 mMCoCl₂, pH 7.9 USER Enzyme (New England BioLabs, Cat# M5505S, 1 U/μL)

Method:

Poly(dA) tailing reactions were performed in 5 iLit reaction volumescontaining either 1×TdT buffer, 0.1 mM dATP, 4 pmol of the substratepolynucleotide 10-01, or 10-127, or 10-128, or 10-129, or 10-139 (or amix of four polynucleotides 10-127, 10-128, 10¬129, and 10-139), and 0or 20 pmol of the attenuator polynucleotide 10-103 and then boiled for 3minutes at 95° C. to ensure that all substrates were single-stranded.Ten units of TdT enzyme were added and the reaction mix was incubated at37° C. for 30 minutes, followed by 10 min incubation at 70° C. toinactivate the TdT enzyme. 0.25 U of the USER enzyme were added andincubated for 5 minutes at 37° C. Samples were boiled in formamideloading buffer and run on a precast 15% TBE-Urea gel (Invitrogen Cat#EC68852BOX), stained with SYBR Gold (Invitrogen Cat #S11494),visualized on a Dark Reader light box (Clare Chemical Research) andphotographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuated poly(dA)tailing reactions by the TDT enzyme are shown in FIG. 18. Lanes 1, 3, 5,7, 9 and 11 show untailed template 10-1001, mixed template (seeMethods), and templates 10-139, 10-127, 10-128 and 10-129, respectively;lanes 2, 4, 6, 8, 10 and 12 show controlled tailed template 10-1001,controlled tailed mixed template, and controlled tailed templates10-139, 10-127, 10-128 and 10¬129, respectively. All of the randomizedpolynucleotide-substrates are tailed similar to the non-random substrate10-001.

Conclusions:

TdT enzyme does not exhibit sequence bias during controlled poly (dA)tailing of single-stranded DNA substrates in the presence of attenuatormolecules.

Example 6 Controlled Poly(dT), Poly(dC), Poly(dG) Tailing ofSingle-Stranded DNA Polynucleotide Template by TdT Enzyme in thePresence of Attenuator Molecules

Materials:

Substrate polynucleotide 10-085 (Table 1) Attenuator polynucleotides:10-103, 10-136, 10-137, and 10-138 (Table 2) 25 bp ladder DNA sizemarker (Invitrogen, Cat #10488-022) TdT Enzyme (New England Biolabs,Cat# M0315S, 20 U/μL) 1 × TDT Buffer: 20 mM Tris-acetate, 50 mMpotassium acetate, 10 mM Mg · acetate, 0.25 mM CoCl₂, pH 7.9 USER Enzyme(New England BioLabs, Cat# M5505S, 1 U/μL)

Method:

Poly(dA), poly(dT), poly(dG) and poly(dC) tailing reactions wereperformed in 5 μL reaction volumes containing 1×TdT buffer, 0.1 mM ofeither dATP, dTTP, dGTP, or dCTP, 4 pmol of the substrate polynucleotide10-185 and 0 or 20 pmol of the attenuator polynucleotide 10-103, 10-136,10-137, or 10-138, respectively. 10 U of the TdT enzyme were added andincubated at 37° C. for 30 minutes, followed by 10 minutes incubation at70° C. to inactivate TdT enzyme. 0.25 U of the USER enzyme were added tothe reaction containing 10-103 and incubated 5 mM at 37° C. Samples wereboiled in formamide loading buffer and run on a precast 15% TBE-Urea gel(Invitrogen Cat #EC68852BOX), stained with SYBR Gold (Invitrogen Cat#S11494), visualized on a Dark Reader light box (Clare ChemicalResearch) and photographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuatedpoly(dA), poly(dT), poly(dG) and poly(dC) tailing reactions by the TDTenzyme are shown in FIG. 19. Lane 1 shows untailed substratepolynucleotide 10-085; lanes 2, 4 6 and 8 show products of controlledtailing of the substrate 10-1085 by dA, dT, dG and dC nucleotides; lanes3, 5 7 and 9 show products of uncontrolled tailing of the substrate10-085 by dA, dT, dG and dC nucleotides; lane 11—25 by ladder DNA sizemarker. Controlled attenuated tailing with dT nucleotides in thepresence of attenuator molecule 10-136 was undistinguishable from thecontrolled attenuated tailing with dA nucleotides in the presence ofattenuator molecule 10-103. Both reactions produced sharp bands with thesize of poly(dA) and poly(dT) tails around 12-13 bases (FIG. 19, lanes 2and 4). Controlled attenuated tailing with dG nucleotides also produceda sharp band with the average size of poly(dG) tail around 10-12 baseswhich agreed with the higher stability of poly(dG/poly(dC) duplex. Theattempt to control poly(dC) tailing was less successful and producedresults that are difficult to interpret.

Conclusions:

Attenuator-controlled poly(dA), poly(dT) and poly(dG) TdT tailingreactions behaved very similarly, resulting in efficient tailing of 100%of templates and adding a very short and accurate homopolymeric tail tothe substrate DNA molecule. Tailing with the dA and the dT nucleotidesproduced tails of about 12-13 bases while tailing with dG nucleotidesproduced tails of about 10-12 bases. Controlled tailing with dCnucleotides was problematic due to difficulty of preparing and handlingof attenuator polynucleotides containing long stretches (greater than 6)of dG-bases (for this reason dT bases were included into the attenuator10-138).

Example 7 Controlled Poly(rA) Tailing of Single-Stranded RNAPolynucleotide Template by the E. coli Poly(A) Polymerase in thePresence of an Attenuator Molecule

Materials:

Substrate RNA polynucleotide 10-191 (Table 1) Attenuator polynucleotide10-103 (Table 2) E. coli poly(A) polymerase (New England Biolabs, Cat#M0276S, 5 U/μL) 1 × Poly(A)polymerase buffer: 50 mM Tris-HCl, 250 mMNaCl, 10 mM MgCl₂, 1 mM ATP, pH 7.9 USER Enzyme (New England Biolabs,Cat# M5505S, 1 U/μL) Low Range ssRNA Marker (New England BioLabs, Cat#N0364S) microRNA Marker: (New England BioLabs, Cat# N2102S)

Method:

Reactions were carried out in a volume of 54, of 1× poly(A) polymerasebuffer containing 4 pmols of substrate polynucleotide 10-191 and 0 or 20pmols of attenuator polynucleotide 10-103, and 2.5 U of poly(A)polymerase. Reactions were performed at 30° C. (FIG. 20) for 5, 10, 15or 30 minutes and then heated to 95° C. to inactivate the poly(A)polymerase. 0.5 U of USER enzyme were then added to the tubes containingattenuator molecules and incubated for 10 minutes at 37° C. Samples werethen boiled in formamide loading buffer and run on a precast 15%TBE-Urea gel (Invitrogen Cat #EC68852BOX), stained with SYBR Gold(Invitrogen Cat #S11494), visualized on a Dark Reader light box (ClareChemical Research) and photographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuated poly(rA)tailing of the RNA substrate polynucleotide 10-191 by poly(A) polymeraseenzyme are shown in FIG. 20. Lanes 4, 5, 6 and 7 and lanes 9, 10, 11 and12 show the tailing kinetics for 5, 10, 15 and 30 minutes of incubationwith the poly(A) polymerase in the absence and in the presence of theattenuator molecule 10-103, respectively. Lanes 1, 2 and 3 show the RNAsubstrate 10-191, the attenuator polynucleotide 10-103 and theattenuator polynucleotide after incubation with the USER enzyme,respectively. Lane 8 shows a combination of the Low Range ssRNA Markerand the microRNA Marker. In both cases the tailing reaction wascompleted within 15 minutes. In the absence of the attenuator moleculethe poly(A) polymerase added to the substrate polynucleotide very longand heterogeneous in size poly(rA) tails. In the presence of theattenuator molecule the size of added poly(rA) tails was substantiallyshorter with a very narrow band corresponding to the substrate with tailof approximately 20 bases (FIG. 20, lane 9). Long attenuator moleculewas degraded by USER enzyme and was not visible on the gel becausedegradation products do not exceed 5 bases (FIG. 20, lanes 2 and 3). Thedimer band, corresponding to the tail size of 40 bases was seen atlonger incubation times (FIG. 20, lanes 10, 11 and 12). Larger size ofcontrolled attenuated tails (20 b) introduced by the E. coli polymerase(A) comparing to tails added by the TdT enzyme (12-13 bases) andappearance of the dimer band in the presence of the same attenuatormolecule 10-103 was explained by a lower thermal stability of thepoly(rA)/poly(dT) duplex versus the poly(dA)/poly(dT) duplex.

Conclusions:

Complete attenuation of poly(rA) tailing of the RNA templates wasachieved using long DNA poly (dT) molecules. The length of poly(rA)tails added by the poly(A) polymerase in the presence of long attenuatormolecules constituted about 20 rA bases with narrow size distributioncontrasting several hundred rA bases added in the absence of attenuatormolecules. Attenuator molecules containing dU bases were degraded aftercompletion of the tailing reaction using USER enzyme to simplifydownstream utilization of the rA-tailed RNA substrates. Attenuatedtailing by poly(A) polymerase produced tails of approximately 20 bases,which can be efficiently used for RNA and microRNA analysis.

Example 8 Controlled Poly(rU) Tailing of Single-Stranded RNAPolynucleotide Template by the Yeast (S. pombe) Poly(U) Polymerase inthe Presence of DNA and RNA Attenuator Molecules

Materials:

Substrate RNA polynucleotide 10-191 (Table 1) Attenuator polynucleotide10-136, 10-192, and 11-049 (Table 2), or High Molecular Weight poly(rA)(Midland Certified Reagent Company, Texas, Catalog # P3001) RNA sizeladder: 0.5 μl Low range ssRNA ladder (NEB N0364S) and 10 μl microRNAmarker (NEB N2102S) combined with 10 μl formamide buffer DNA sizeladder: 25 bp ladder DNA size marker (Invitrogen, Cat #10488-022) Yeast(S. pombe) poly(U) polymerase (New England Biolabs Cat# M0337S, 2 U/μL)1 × Poly(U)polymerase buffer: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂, 1mM UTP, 1 mM DTT, pH 7.9 Formamide buffer: 97% Formamide, 10 mM EDTA,0.01% bromophenol blue, 0.01% xylene cyanol

Method:

Reactions were carried out in a volume of 5 uL of 1× poly(U) polymerasebuffer containing 4 picomoles (pmols) of substrate polynucleotide 10-191and 0 or 20 pmols of the DNA attenuator polynucleotides 10-136 and10-192 or High Molecular Weight (HMW) RNA attenuator poly(rA) (averagesize about 200 b). Reactions were performed at either 37° C. (FIG. 21A)or 30° C. (FIG. 21B) for 10, 15 or 30 minutes. Reactions with RNAattenuator were prepared by adding 8 pmols of the substrateribo-polynucleotide 10-191 and either 40 pmols of ribo-polynucleotide11-049 or 41 ultrapure water to reaction tubes containing 1×NEB 2 bufferand 1 mM rUTP. To each tube, 2 U of NEB poly(U) polymerase was added andthe reactions incubated at 30° C. (FIG. 7C) for 15 minutes. To stop thereactions, 10 ml of 2× formamide loading buffer was added to each tube,boiled and run on a precast 15% TBE-Urea gel (Invitrogen Cat#EC68852BOX), stained with SYBR Gold (Invitrogen Cat #S11494),visualized on a Dark Reader light box (Clare Chemical Research), andphotographed using a digital camera.

Results:

Electrophoretic analysis of products of standard and attenuated poly(rU)tailing reactions by poly(U) polymerase enzyme are shown in FIG. 21a andFIG. 21b . FIG. 21a shows the time course of controlled (lanes 6, 7 and8) and uncontrolled (lanes 3, 4 and 5) poly(U) tailing of the RNAsubstrate 10-191 for 10, 15 and 30 minute incubation times,respectively, in the presence of relatively short DNA attenuatorpolynucleotide 10-136 (20 b); lane 2—original RNA substrate 10-191; lane1-25 bp ladder DNA size marker. FIG. 21b shows controlled tailing of theRNA substrate 10-191 (FIG. 21b , lane 2) in the presence of long 40 baseDNA attenuator (FIG. 21b , lane 3) and High Molecular Weight poly(rA)RNA attenuator (FIG. 21b , lane 4). Lane 1 shows a combination of theLow Range ssRNA Marker and the microRNA Marker, lane 5—a mixture of theRNA substrate 10-191 and High Molecular Weight poly(rA) RNA attenuator.FIG. 20C shows controlled tailing of the RNA substrate 10-191 (lane 3)in the presence of long 30b RNA attenuator polynucleotide 11-049. As wasseen from FIG. 21a , attenuated tailing in the presence of short DNApoly(dA)20 (SEQ ID NO: 21) attenuator resulted in a repetitive tailingpattern (ladder) that is indicative of the attenuation process workingbut the complete inhibition by 20-base DNA attenuator and narrow tailsize distribution can't be achieved. Increasing the length of the DNAattenuator to 40 bases did improve the attenuation process and resultedin a single band that was broad (FIG. 21b , lane 3). It is known thatpoly(rU) and poly(dA) polymers form very unstable duplexes, whilepoly(rU) and poly(rA) form much more stable complexes. This wasconfirmed by using both High Molecular Weight poly(rA) and shorterribo-polynucleotide (rA)30 (SEQ ID NO: 25) (11-049) as an attenuator forthe poly(U) RNA tailing. As can be seen from FIG. 21b , lane 4, thetailing in the presence of long RNA attenuator resulted in a tailingproduct with very narrow size distribution and a tail size of about 20bases (19 bases of substrate plus approximately 20 bases of poly(U) tailproduce a molecule with about a size of 40 bases, similar to the size ofthe 40 base attenuator 10-192). Similar results were obtained usingattenuator ribo-polynucleotide 11-049 (FIG. 21c lane 3) wherepoly(U)-tailed substrate ribo-polynucleotide 10-191 can be seen as aproduct of about 40 bases, suggesting that the size of the poly(U) tailis about 20 bases.

Conclusions:

Controlled attenuated poly(U) tailing was achieved in the presence ofDNA poly(dA) attenuators but it is much more efficient in the presenceof RNA poly(rA) attenuators. Attenuated tailing by poly(U) polymeraseproduced tails of approximately 20 bases that are efficiently used forRNA and microRNA analysis.

Example 9 Simultaneous Controlled Poly(dA) Tailing andAttenuator-Adaptor Molecule Ligation to Single-Stranded DNA by CombinedAction of TdT and E. coli DNA Ligase Enzymes

Materials:

Substrate polynucleotide 10-105 (Table 1) Double strandedattenuator-adaptor formed by polynucleotides 10-211 and 10-212 (Table 3and Table 6) TdT Enzyme (New England Biolabs, Cat# M0315S, 20 U/μL) 1 ×TDT Buffer: 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Mg ·acetate, 0.25 mM CoCl₂, pH 7.9 E. coli DNA Ligase Enzyme (New EnglandBioLabs, Cat# M0205S, 10 U/μL)

Method:

The polynucleotides 10-211 and 10-212 were annealed together by boilingand then allowed to slowly cool to room temperature in 10 mM Tris-HClcontaining 0.1 mM EDTA and 50 mM NaCl. Simultaneous poly(dA) tailing andattenuator-adaptor ligation reactions were performed in a 10 μL reactionvolume containing 1×TdT buffer, 0.1 mM dATP, 26 uM NAD+, 4 pmol of thesubstrate polynucleotide and 20 pmol of the attenuator-adaptor molecule.10 U TdT enzyme and either 0 or 10 U DNA ligase enzyme were added andincubated at 37° C. for 15 minutes. Samples were then boiled informamide loading buffer and run on a precast 15% TBE-Urea gel(Invitrogen Cat #EC68852BOX), stained with SYBR Gold (Invitrogen Cat#S11494), visualized on a Dark Reader light box (Clare ChemicalResearch) and photographed using a digital camera.

Results:

Electrophoretic analysis of products of simultaneously attenuatedTdT-mediated poly(dA) tailing and adaptor ligation reaction are shown inFIG. 22. Lane 1 represents the 25 bp ladder DNA marker, lane 2—thesubstrate and attenuator-adaptor molecules used in the reaction. Lane 3shows the products of simultaneous tailing-ligation reaction, lane 4—theproducts of tailing reaction using attenuator-adaptor construct (noligase). In the presence of TdT enzyme and E. coli ligase, the reactionresulted in a sharp band that is located between 75 and 100 base bandsof the 25 base DNA ladder. The expected size of tailing and ligationproduct is 95 bases, which is very close to the size of the observedproduct (FIG. 22, lane 3, the largest band). The 50 base bandcorresponding to the non-reacted substrate is almost not visible in lane4, indicating that efficiency of the attenuated tailing-ligationreaction is close to 100%. Data presented in lanes 4 (TdT only) and 5(TdT and ligase) suggested that the reaction time used (15 minutes) issufficient for adding a 12 base tail and attaching the adaptor but notsufficient to convert the substrate into a product with 12-13 added dAbases (SEQ ID NO: 86). Schematically the single-tube, single-step DNAtailing-ligation process is shown on FIG. 24 a.

Conclusions:

Poly(dA) tailing and subsequent ligation to the attenuator-adaptormolecule happened very quickly and efficiently when performed inparallel in a single sample tube. The reaction is used for efficientadaptation or tagging of random single-stranded DNA molecules in asingle-tube, single-reaction format.

Example 10 Simultaneous Controlled Poly(dA) Tailing and Immobilizationof Single-Stranded DNA by Combined Action of the TdT and DNA LigaseEnzymes and Use of Attenuator-Adaptors Immobilized to Magnetic Beads

Materials:

Substrate polynucleotide 10-105 (Table 1): Attenuator-adaptor formed bypolynucleotides 10-211 and 10-212 (Table 3 and Table 6) TdT Enzyme (NewEngland Biolabs, Cat# M0315S, 20 U/μL) 1 × TDT Buffer: 20 mMTris-acetate, 50 mM potassium acetate, 10 mM Mg · acetate, 0.25 mMCoCl₂, pH 7.9 E. coli DNA Ligase (New England BioLabs, Cat# M0205S, 10U/μL) Dynabeads MyOne Streptavidin T1 (Invitrogen Cat#656.01) Bead WashBuffer: 5 mM Tris-HCl ph 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20

Method:

The attenuator-adaptor complex was prepared as described in Example 9.100 μL of Dynabeads were washed twice with bead wash buffer and thenresuspended in 20 bead wash buffer. To the bead solution, 80 pmols of10-211/212 annealed pair was added. Beads were incubated at roomtemperature on the orbital shaker (Nutator) for approximately 2 hours,then stored at 4° C. until needed. Immediately before running reactions,54, of bead solution was transferred to a new tube, and washed twicewith TdT buffer. Simultaneous poly(dA) tailing and attenuator ligationreactions were performed in 10 μL reaction volumes containing 1×TdTbuffer, 0.1 mM dATP, 26 μM NAD+, 4 pmol of the substrate polynucleotideand 20 pmol of the attenuator-adaptor complex immobilized on the beads.10 U TdT enzyme and either 0 or 10 U DNA ligase enzyme were added andincubated at 37° C. for 15 minutes. The beads were washed twice withdeionized water and then with 10 μL of 125 mM NaOH to stripnon-biotinylated ssDNA from the beads. DNA released by NaOH wasneutralized and the remaining samples were then boiled in formamideloading buffer and run on a precast 15% TBE¬Urea gel (Invitrogen Cat#EC68852BOX), stained with SYBR Gold (Invitrogen Cat #S11494),visualized on a Dark Reader light box (Clare Chemical Research) andphotographed using a digital camera.

Results:

Electrophoretic analysis of products of simultaneously attenuatedTdT-mediated poly(dA) tailing and ligation of the immobilizedattenuator-adaptor are shown in FIG. 22, lane 5. The expected size oftailing and ligation product is 95 bases, which is very close to thesize of the observed product (FIG. 22, lane 5, the largest band) and theproduct of tailing-ligation reaction described in Example 9 (lane 3).Intensities of 95 base pair bands in lanes 3 and 5 indicated thatefficiency of the attenuated tailing-ligation-immobilization reactionwas close to 100%. The strong band corresponding to polynucleotide10-212 in lane 5 was due to the non-reacted adaptor which is present inexcess. The immobilization process is shown in FIG. 24 b.

Conclusions:

Poly(dA) tailing and subsequent ligation to the immobilizedattenuator-adaptor molecule happened very quickly and efficiently whenperformed in parallel in a single sample tube. The reaction is used forefficient adaptation, tagging and immobilization of randomsingle-stranded DNA molecules in a single-tube, single-reaction format.

Example 11 Simultaneous Controlled Poly(rA) Tailing andAttenuator-Adaptor Molecule Ligation to Single-Stranded RNA by CombinedAction of the Yeast Poly(A) Polymerase and T4 DNA Ligase Enzymes

Materials:

Substrate polynucleotide 10-191 (Table 1): Attenuator-adaptor formed bypolynucleotides 11-010 and 11-011 (Table 3 and Table 6) Yeast Poly(A)Polymerase (Affymetrix, 74225Y: 600 U/μl) 1 × TDT Buffer: 20 mMTris-acetate, 50 mM potassium acetate, 10 mM Mg · acetate, 0.25 mMCoCl₂, pH 7.9 T4 DNA Ligase (New England BioLabs, Cat# M0202T,2,000,0000 end units/ml Dynabeads MyOne Streptavidin T1 (InvitrogenCat#656.01) Bead Wash Buffer: 5 mM Tris-HCl ph 7.5, 0.5 mM EDTA, 1MNaCl, 0.05% Tween-20 5X Poly(A) Polymerase Reaction Buffer, (Affymetrix:74226): 100 mM Tris-HCl pH 7.0, 3 mM MnCl₂, 0.1 mM EDTA, 1 mMDithiothreitol, 500 ug/mL acetylated BSA, 50% Glycerol TrackIt 25 bpLadder: Invitrogen: 10488-022 Formamide Buffer: 97% Formamide, 10 mMEDTA, 0.01% bromophenol blue, 0.01% xylene cyanol (made in-house)

Method:

Reactions were prepared by adding 8 pmols of the substrateribo¬oligonucleotide (10-191) and 40 pmols of attenuator/adaptor oligopair (11-010/11-011) to reaction tubes containing 1×Poly(A) Polymerasereaction buffer and 1 mM rATP. To each tube 300 units of yeast poly(A)polymerase and 2,000 cohesive end units of T4 DNA ligase were added andthe reactions incubated at 37° C. for 30 minutes. To stop the reaction10 μL of 2× Formamide loading buffer was added to each tube and thereactions were boiled at 95° C. for 2 minutes. A 15% TBE-Urea gel wasloaded with 25 by Ladder and 10 μL of each reaction. A current of 200volts was applied to the gel for 30 minutes to separate the moleculesand the gel was then stained with SYBR Gold for 10 minutes and thenvisualized on a Dark Reader light box (Clare Chemical Research) andphotographed using a digital camera.

Results:

Electrophoretic analysis of products of simultaneously tailed andligated substrate by Poly(A) polymerase and T4 DNA Ligase enzymes areshown in FIG. 23. Schematically the single-tube, single-step RNAtailing-ligation process is shown on FIG. 24 c.

Conclusion:

A synthetic RNA substrate can have a DNA adaptor sequence ligated to the3′ end of the RNA substrate by combined attenuated poly (rA)-tailing andligation catalyzed by poly(A) polymerase and T4 DNA ligase. The reactioncan be used for efficient adaptation and tagging of randomsingle-stranded RNA molecules in a single-tube, single-reaction format.

TABLE 1 Synthetic polynucleotide substrates ID SEQ ID NO Sequence10-001:  1 5′-GGT CGT AGC AGT CGT TGA TG-3′ 10-105:  25′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CTT TGG ACA CGG GG-3′10-106:  3 5′-CCC CGT GTC CAA AGG TGC GTT TAT AGA TCT AGATCT AGA CTA GGT TGC AGC AAC TA-3′ Phosphate 10-107:  45′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CTT TGG ACA CTT TT-3′10-108:  5 5′-AAA AGT GTC CAA AGG TGC GTT TAT AGA TCT AGACTA GGT TGC AGC AAC TA-3′ Phosphate 10-109:  65′-CGT GTC CAA AGG TGC GTT TAT AGA TCT AGA TCTAGA TCT AGA CTA GGT TGC AGC AAC TA-3′ Phosphate 10-110:  75′-GGT CCC CGT GTC CAA AGG TGC GTT TAT AGA TCTAGA TCCT AGA TCT AGA CTA GGT TGC AGC AAC TA-3′ Phosphate-3 10-085:  85′-GTA TCG CTA CGT TGT CAC ACA CTA CAC TGC TCG ACA GTA AAT ATG CCA AT-3′10-127:  9 5′-GAT CGT AGC TAG (N)₁₁ G-3′ 10-128: 105′-GAT CGT AGC TAG (N)₁₁ C-3′ 10-129: 11 5′-GAT CGT AGC TAG (N)₁₁ T-3′10-139: 12 5′-GAT CGT AGC TAG (N)₁₁ A-3′ 10-191: 135′-rGrGrCrCrUrUrGrUrUrCrCrUrGrUrCrCCrCrA-3′

TABLE 2 Synthetic polynucleotide-attenuators ID SEQ ID NO Sequence10-103: 14 5′-(TTTTTU₆TTTT-3′Phosphate 10-130: 155′-GAT CGT AU T₇ rUrU-3′ 10-131: 16 5′-GAT CGT AU T₈ rUrU-3′ 10-132: 175′-GAT CGT AU T₉ rUrU-3′ 10-133: 18 5′-GAT CGT AU T₁₀ rUrU-3′ 10-134: 195′-GAT CGT AU T₃₁ rUrU-3′ 10-135: 20 5′-GAT CGT AU T₁₂ rUrU-3′ 10-136:21 5′-(AAA)₆AA-3′Phosphate 10-137: 22 5′-(CCC)₅CC-3′Phosphate 10-138: 235′-(GGG GGT)₆GGG G3′Phosphate 10-192: 24 5′-(AAA)₁₀A-3′Phosphate 11-04925 5′-(rArArA)₁₀-3′Phosphate

TABLE 3Synthetic polynucleotides comprising the attenuator-adaptor complex IDSEQ ID NO Sequence 10-211 265′ Biotin-TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGATC TTT TTT TTT TrUrU-3′ 10-212 275′ Phosphate-GAT CGG AAG AGC GTC GTG TAG GGA AAG AG GTA-3′ Phosphate11-010 28 5′ Phosphate-CTT ATT GCT GTG GTT GGT TCC TGT GCTGTT TT-3′ Phosphate 11-011 29 5′-CCAACCACAGCAAUAAGUTTTUTTTTUTTTTUTTTT-3′Phosphate

TABLE 4 Synthetic polynucleotides comprising the DNA tailing size markerID SEQ ID NO Sequence 10-001 30 5′-GGT CGT AGC AGT CGT TGA TG-3′ 10-00931 5′-GGT CGT AGC AGT CGT TGA TGA AAAA-3′ 10-100 325′-GGT CGT AGC AGT CGT TGA TGA AAA AAA AAA-3′ 10-101 335′-GGT CGT AGC AGT CGT TGA TGA AAA AAA AAA AAA AA-3′ 10-102 345′-GGT CGT AGC AGT CGT TGA TGA AAA AAA AAA AAA AAA AAAA-3′

TABLE 5 Double stranded DNA substrates ID SEQ ID NO SequenceBlunt end, GC-Rich 10-105 355′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CCTT TGG ACA CGGGG-3′10-106 36 3P′-ATC AAC GAC GTT GGA TCA GAT CTA GAT ATT TGCGTG GAA ACC TGT GCC CC-5′ Blunt end AT-Rich 10-107 375′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CTT TGG ACA CTT TT-3′10-108 38 3P′-ATC AAC GAC GTT GGA TCA GAT CTA GAT ATT TGCGTG GAA ACC TGT GAAAA-5′ 3′-Overhang End (3 bases) 10-105 355′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CTT TGG ACA CGGGG-3′10-109 39 3P′-ATC AAC GAC GTT GGA TCA GAT CTA GAT ATT TGCGTG GAA ACC TGT GC-5′ 3′-Recessed Endd (3 bases) 10-105 355′-TAG TTG CTG CAA CCT AGT CTA GAT CTA TAA ACG CAC CTT TGG ACA CGGGG-3′10-110 40 3P′-ATC AAC GAC GTT GGA TCA GAT CTA GAT ATT TGCGTG GAA ACC TGT GCC CCTGG-5′ Attenuator-adaptor 10-212 415′P-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA-P 10-211 423′-rUrUTTTTTTTTTTCTAGCC TTCT CGCAGCACATCC CTTT CTCACAT-Biotin-5′

TABLE 6 Structure of the Attenuator-Adaptor Complex ID SEQ ID NOStructure 10-212 10-211 41 42

11-010 11-011 28 29

The following Table 7 provides NGS Adaptor Sequences corresponding toNGS sequence X and sequence Y (see FIGS. 25-29).

Adaptor Sequence (5′-3′) SEQ ID NO Ion Torrent Adaptor ACCATCTCATCCCTGCGTGTCTCCGACTCAG 44 Ion Torrent P1CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 45 Illumina Adaptor 1P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG 46 Illumina Adaptor 2 ACACTCTTTCCCTACACGACGCTCTTCCGATCT 47 Roche 454 Adaptor ACCATCTCATCCCTGCGTGTCTCCGACTCAG 48 Roche 454 Adaptor bCCTATCCCCTGTGTGCCTTGGCAGTCTCAG 49 SOLID Adaptor P1CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 50 SOLID Adaptor P2AGAGAATGAGGAACCCGGGGCAGTT 51

Example 12: Controlled Tailing and Ligation Reaction withAttenuator-Adaptor Molecules

Materials:

10 μM Adaptor oligonucleotide 13-128 (Table X) 10 μM Attenuator-adaptoroligonucleotide 7N 13-281 10 μM Attenuator-adaptor oligonucleotide 6N13-280 10 μM Attenuator-adaptor oligonucleotide 5N 13-279 10 μMAttenuator-adaptor oligonucleotide 3N 13-278 Substrate oligonucleotide12-492 T4 DNA ligase (Rapid) 600,000 U/ml (Enzymatics, Cat# L6030-HC-L)Terminal deoxynucleotidyl transferase 20,000 U/ml (Enzymatics, Cat#P7070L) 10X Green Buffer (Enzymatics, Cat# B0120) Adenosine5′-Triphosphate (ATP) 10 mM (New England BioLabs, Cat# P0756S) 100 mMdATP Set (Life technologies, Invitrogen Cat# 10216-018) 25 bp ladder DNAsize marker (Life technologies, Invitrogen Cat# 10488-022)

Method:

A controlled tailing and ligation reaction was assembled in this orderin a total volume of 40 μl at a final concentration of 0.25 μM ofSubstrate oligonucleotide 12-492, 0.75 μM Adaptor oligonucleotide13-128, 1.5 μM Attenuator-adaptor oligonucleotide 13-281 or 1.5 μMAttenuator-adaptor oligonucleotide 13-280 or 1.5 μM Attenuator-adaptoroligonucleotide 13-279 or 1.5 μM Attenuator-adaptor oligonucleotide13-278 or a combination of these four with 0.375 μM of each, or 0.750 μMof each, or 1.125 μM of each, 1× Green Buffer, 1 mM ATP, 1 mM dATP, 15U/[μl T4 DNA ligase and 0.5 U/μl Terminal deoxynucleotidyl transferase.

The reaction was incubated at 25° C. for 10 minutes, 95° C. for 2minutes. Next, 10[il of the sample were boiled with 2× formamide loadingbuffer and subsequently run on a pre-casted 15% polyacrylamide gel,TBE-Urea (Invitrogen, Cat #S11494), stained SYBR® Gold nucleic acid gelstain (Invitrogen, Cat #S11494), visualized on a Dark reader light box(Clare Chemical Research) and photographed using a digital camera.

Results:

Controlled tailing and ligation reactions were performed and visualizedon a 15% polyacrylamide gel by electrophoresis under denaturingconditions. Gel 1 lane 1 contains only oligonucleotides. Gel 1 lane 2,3, and 4 show a band representing the product of addition of thehomopolymer (approximately 2-6 base pairs) and ligation of the adaptor(23 base pairs) to the substrate (43 base pairs) (FIG. 34). Gel 2 lane1, 2, 3, and 4 shows a band representing the product of addition of thehomopolymer (approximately 2-6 base pairs) and ligation of the adaptor(23 base pairs) to the substrate (43 base pairs) (FIG. 35). Bandscorresponding to the product of addition of a homopolymer tail andligation to the target substrate are observed in the presence of therandom base attenuator-adaptors 13-278, 13-279, 13-280, 13-281, and anequimolar combination of all four random base attenuator-adaptors.

Conclusion:

The addition of a homopolymer tail and ligation to the target substratewas accomplished with random base attenuator-adaptors 13-278, 13-279,13-280, and 13-281 to varying efficiencies.

Example 13. Controlled Tailing and Ligation Reaction with DinucleotideAttenuator-Adaptors

Materials:

Substrate oligonucleotide (12-492) Adaptor oligonucleotide (13-128)Attenuator-adaptor oligonucleotide 12T (13-114) Attenuator-adaptoroligonucleotide 6C (13-263) Attenuator-adaptor oligonucleotide 6K(13-274) where K corresponds to G/T dinucleotide Attenuator-adaptoroligonucleotide 6R (13-275) where R corresponds to G/A dinucleotide T4DNA ligase (Rapid) 600,000 U/ml (Enzymatics, Cat# L6030-HC-L) Terminaldeoxynucleotidyl transferase 20,000 U/ml (Enzymatics, Cat# P7070L) 10XGreen Buffer (Enzymatics, Cat# B0120) Adenosine 5′-Triphosphate (ATP) 10mM (New England BioLabs, Cat# P0756S) 100 mM deoxyribonucleosidetriphosphates (dNTP) Set (Life technologies, Invitrogen Cat# 10297-117)25 bp ladder DNA size marker (Life technologies, Invitrogen Cat#10488-022)

Method:

A controlled tailing and ligation reaction was assembled in this orderin a total volume of 40 μl at a final concentration of 0.25 μM ofSubstrate oligonucleotide 13-325, 0.75 μM Adaptor oligonucleotide13-128, 1.5 μM Attenuator-adaptor oligonucleotides with attenuatorportions corresponding to 12T or 6C homopolymers or a plurality of 6R(G/A) or 6K (G/T) randomly synthesized dinucleotides, 1× Green Buffer,0.5 mM, 1 mM ATP, 1 mM of appropriate dNTP mononucleotide ordinucleotide mixture complementary to the mononucleotide or dinucleotideattenuator-adaptor used (see gel label), 15 U/μl T4 DNA ligase and 0.5U/μl Terminal deoxynucleotidyl transferase.

The reaction was incubated at 25° C. for 30 minutes, followed byincubation at 95° C. for 2 minutes. Next, 10 μl of the sample wereboiled with formamide loading buffer 2× and subsequently run on apre-casted 15% polyacrylamide gel, TBE-Urea (Invitrogen, Cat #S11494),stained SYBR® Gold nucleic acid gel stain (Invitrogen, Cat #S11494),visualized on a Dark reader light box (Clare Chemical Research) andphotographed using a digital camera.

Results:

Controlled tailing and ligation reactions were performed and visualizedon a 15% polyacrylamide gel by electrophoresis under denaturingconditions. Lanes 2, 3, 4 and 5 show the tailed and ligated product justbelow 75 base marker, corresponding to the ligation of the 23 basesadaptor (13-128) to the 43 bases substrate (12-492) which was tailed bythe TdT enzyme (approximately 6 bases) for a product size about 72bases. Adaptor (13-128) and attenuator¬adaptor excess are also observed.Some leftover product is also observed at 43 bases in lane 2,4 and 5.Lane 1 corresponds to the DNA polynucleotide marker spiked withSubstrate oligonucleotide (12-492), Adaptor oligonucleotide (13-128) andAttenuator-adaptor oligonucleotide 6K (13-274) (FIG. 36).

Conclusions:

Controlled tailing and ligation reactions are efficient usingdinucleotide tailing with the corresponding complementary plurality ofrandom based dinucleotide attenuator-adaptors.

TABLE 8 Synthetic polynucleotide substrate for Examples 12 and 13 SEQ IDID NO Sequence 12-492 52 /5PHOS/NNNNNNNNNNTGCCTCCTGGACTATGTCCGGGTANNNNNNNNNN

TABLE 9Synthetic polynucleotide attenuator-adaptors for Examples 12 and 13 IDSEQ ID NO Sequece 13-281 53 CAGTCGGUGATTNNNNNNN/3SpC3/ 13-280 54CAGTCGGUGATTTNNNNNN/3SpC3/ 13-279 55 CAGTCGGUGATTTTNNNNN/3SpC3/ 13-27856 CAGTCGGUGATTTTTTNNN/3SpC3/ 13-114 57 CAGTCGGTGAUTTTTTUTTTTTT/3SpCS3/13-263 58 CAGTCGGUGATCCCCCC/3SpC3/ 13-274 59 CAGTCGGUGATKKKKKK/3SpC3/13-275 60 CAGTCGGUGATRRRRRR/3SpC3/

TABLE 10 Synthetic polynucleotides comprising the adaptorfor Examples 12 and 13. SEQ ID ID NO Sequence 13-128 61/5Phos/ATCACCGACTGCCCATAGAGAGG/3Phos/

The invention claimed is:
 1. A method of attenuated tailing of asubstrate polynucleotide comprising: (i) adding (1) atemplate-independent nucleic acid polymerase, (2) an attenuatorpolynucleotide comprising an attenuator sequence, (3) nucleotidescomplementary to the attenuator sequence, (4) a first adaptorpolynucleotide and (5) a ligase to a sample comprising the substratepolynucleotide thereby yielding a first reaction mixture, wherein theattenuator sequence is from about 10 nucleotides to about 100nucleotides in length, wherein the attenuator polynucleotide furthercomprises a sequence W positioned adjacent to the attenuator sequence,wherein the attenuator polynucleotide comprises a 3′ blocking group, andwherein the first adaptor polynucleotide comprises a sequence X which iscomplementary to sequence W of the attenuator polynucleotide; and (ii)incubating the first reaction mixture under conditions sufficient toallow (1) the template-independent nucleic acid polymerase to add a tailsequence to the 3′ end of the substrate polynucleotide, (2) theattenuator sequence to hybridize with the tail sequence, and (3)ligation of the first adaptor polynucleotide to the substratepolynucleotide to yield a single adaptor substrate polynucleotide. 2.The method of claim 1, further comprising: (iii) adding a primer, apolymerase and deoxynucleotides to the first reaction mixture followingstep (ii) to form a second reaction mixture, wherein the primer iscomplementary to at least a portion of sequence X; (iv) incubating thesecond reaction mixture under conditions sufficient to performpolymerase extension from the primer thereby producing a second strandpolynucleotide with sequence complementary to the single adaptorsubstrate polynucleotide; (v) adding a second adaptor polynucleotide anda ligase to the second reaction mixture following step (iv) to form athird reaction mixture; and (vi) incubating the third reaction mixtureunder conditions sufficient to ligate the second adaptor polynucleotideto the single adaptor substrate polynucleotide.
 3. The method of claim2, further comprising (vii) isolating the second strand polynucleotideand single adaptor substrate polynucleotide from the second reactionmixture to yield a purified nucleic acid mixture, wherein step (vii) isperformed between step (iv) step (v), and wherein the second reactionmixture of step (v) is the purified nucleic acid mixture.
 4. The methodof claim 3, wherein the second adaptor polynucleotide comprises asequence Y and a sequence V, wherein sequence V is complementary tosequence Y when sequence V is the same length as sequence Y, or whereinsequence V is complementary to a portion of sequence Y when sequence Vis less than the length of sequence Y, and wherein the second adaptorpolynucleotide is a separate molecule from the attenuatorpolynucleotide.
 5. The method of claim 1, wherein the substratepolynucleotide is selected from the group consisting of asingle-stranded substrate polynucleotide, a double-stranded substratepolynucleotide, a partially double-stranded substrate polynucleotide, abisulfite-treated substrate polynucleotide, a product of a primerextension reaction, and cDNA.
 6. The method of claim 1, wherein theattenuator sequence is a homopolymeric sequence selected from the groupconsisting of poly (dA), poly (dT), poly (dC), poly (dG), and poly (dU).7. The method of claim 1, wherein the attenuator sequence is ahomopolymeric sequence selected from the group consisting of poly (rA),poly (U), poly (rC), and poly (rG).
 8. The method of claim 1, whereinthe attenuator sequence is a heteropolymeric sequence selected from thegroup consisting of dA and rA bases; dT, dU and U bases; dC and rCbases; and dG and rG bases.
 9. The method of claim 1, wherein theattenuator sequence is a dinucleotide sequence selected from the groupconsisting of dG and dC, dA and dT, dG and dT, dG and dA, dA and dC, anddC and dT.
 10. The method of claim 1, wherein the template-independentnucleic acid polymerase is terminal deoxynucleotidyl transferase (TdT).11. The method of claim 1, wherein the template-independent nucleic acidpolymerase is an RNA-specific nucleotidyl transferase selected from thegroup consisting of poly(A) polymerase and poly(U) polymerase.
 12. Themethod of claim 1, wherein the addition of the tail sequence to thesubstrate polynucleotide is temperature-sensitive, and wherein the tailsequence is added at a temperature selected from 4° C. to 50° C.
 13. Themethod of claim 1, wherein the conditions in step (ii) are sufficient toallow the addition of at least 10 nucleotides to the substratepolynucleotide.
 14. The method of claim 1, wherein the substratepolynucleotide is a single-stranded DNA or RNA.
 15. The method of claim1, wherein the substrate polynucleotide is immobilized after step (ii).